http://journals.iucr.org/d/journalhomepage.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. en-gb Copyright (c) 2013 International Union of Crystallography International Union of Crystallography International Union of Crystallography http://journals.iucr.org urn:issn:0907-4449 Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. text/html Open access article in Acta Crystallographica Section D Biological Crystallography text monthly 1 2002-01-01T00:00+00:00 med@iucr.org Acta Crystallographica Section D Biological Crystallography Copyright (c) 2013 International Union of Crystallography urn:issn:0907-4449 http://journals.iucr.org/logos/rss10d.gif http://journals.iucr.org/d/journalhomepage.html Still image http://scripts.iucr.org/cgi-bin/paper?mn9017 The two structures reported in the article by Chin et al. [(2013). Acta Cryst. D69, 352–366] have been further refined and corrected. Copyright (c) 2013 International Union of Crystallography urn:issn:0907-4449 Chin, K.-H. Tu, Z.-L. Su, Y.-C. Yu, Y.-J. Chen, H.-C. Lo, Y.-C. Chen, C.-P. Barber, G.N. Chuah, M.L.-C. Liang, Z.-X. Chou, S.-H. 2013-06-01 doi:10.1107/S0907444913012158 International Union of Crystallography The article by Chin et al. [(2013). Acta Cryst. D69, 532–366] is corrected. en MOUSE STING; C-DI-GMP; INNATE IMMUNE RECEPTORS; [PI]-HELIX; CORRIGENDUM The two structures reported in the article by Chin et al. [(2013). Acta Cryst. D69, 352–366] have been further refined and corrected. text/html Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING. Corrigendum text 6 69 2013-06-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2013 International Union of Crystallography 0907-4449 addenda and errata 1191 med@iucr.org 1191 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5090 Research projects in structural biology increasingly rely on combinations of heterogeneous sources of information, e.g. evolutionary information from multiple sequence alignments, experimental evidence in the form of density maps and proximity constraints from proteomics experiments. The OpenStructure software framework, which allows the seamless integration of information of different origin, has previously been introduced. The software consists of C++ libraries which are fully accessible from the Python programming language. Additionally, the framework provides a sophisticated graphics module that interactively displays molecular structures and density maps in three dimensions. In this work, the latest developments in the OpenStructure framework are outlined. The extensive capabilities of the framework will be illustrated using short code examples that show how information from molecular-structure coordinates can be combined with sequence data and/or density maps. The framework has been released under the LGPL version 3 license and is available for download from http://www.openstructure.org. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Biasini, M. Schmidt, T. Bienert, S. Mariani, V. Studer, G. Haas, J. Johner, N. Schenk, A.D. Philippsen, A. Schwede, T. 2013-05-01 doi:10.1107/S0907444913007051 International Union of Crystallography Current developments in the computational structural biology framework OpenStructure are presented. en OPENSTRUCTURE; COMPUTATIONAL STRUCTURAL BIOLOGY Research projects in structural biology increasingly rely on combinations of heterogeneous sources of information, e.g. evolutionary information from multiple sequence alignments, experimental evidence in the form of density maps and proximity constraints from proteomics experiments. The OpenStructure software framework, which allows the seamless integration of information of different origin, has previously been introduced. The software consists of C++ libraries which are fully accessible from the Python programming language. Additionally, the framework provides a sophisticated graphics module that interactively displays molecular structures and density maps in three dimensions. In this work, the latest developments in the OpenStructure framework are outlined. The extensive capabilities of the framework will be illustrated using short code examples that show how information from molecular-structure coordinates can be combined with sequence data and/or density maps. The framework has been released under the LGPL version 3 license and is available for download from http://www.openstructure.org. text/html OpenStructure: an integrated software framework for computational structural biology text 5 69 2013-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 701 med@iucr.org 709 1399-0047 http://scripts.iucr.org/cgi-bin/paper?en5538 Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Simonetti, A. Marzi, S. Fabbretti, A. Hazemann, I. Jenner, L. Urzhumtsev, A. Gualerzi, C.O. Klaholz, B.P. 2013-06-01 doi:10.1107/S0907444913006422 International Union of Crystallography The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. en TRANSLATION INITIATION FACTOR 2; THERMUS THERMOPHILUS; GTP; GDP Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue. text/html Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms text 6 69 2013-06-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers med@iucr.org 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5227 Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo5U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo5U and was annotated as an S-adenosyl-l-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-l-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxy­methyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-­ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Byrne, R.T. Whelan, F. Aller, P. Bird, L.E. Dowle, A. Lobley, C.M.C. Reddivari, Y. Nettleship, J.E. Owens, R.J. Antson, A.A. Waterman, D.G. 2013-06-01 doi:10.1107/S0907444913004939 International Union of Crystallography The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group. en SCM-SAH; ESCHERICHIA COLI; PUTATIVE TRNA-MODIFICATION ENZYME; CMO5U BIOSYNTHESIS Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo5U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo5U and was annotated as an S-adenosyl-l-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-l-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxy­methyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-­ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM. text/html S-Adenosyl-S-carboxymethyl-l-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA text 6 69 2013-06-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 1090 med@iucr.org 1098 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mn5025 The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Squires, G. Pous, J. Agirre, J. Rozas-Dennis, G.S. Costabel, M.D. Marti, G.A. Navaza, J. Bressanelli, S. Guerin, D.M.A. Rey, F.A. 2013-06-01 doi:10.1107/S0907444913004617 International Union of Crystallography The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV. en TRIATOMA VIRUS; TRV; CRIPAVIRUS; DICISTROVIRIDAE; VIRUS STRUCTURE; X-RAY CRYSTALLOGRAPHY The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed. text/html Structure of the Triatoma virus capsid text 6 69 2013-06-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 1026 med@iucr.org 1037 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5199 Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central β-sheet that connects the N-terminal and C-­terminal domains. In bovine chymosin the N-terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate-binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Langholm Jensen, J. Mølgaard, A. Navarro Poulsen, J.-C. Harboe, M.K. Simonsen, J.B. Lorentzen, A.M. Hjernø, K. van den Brink, J.M. Qvist, K.B. Larsen, S. 2013-05-01 doi:10.1107/S0907444913003260 International Union of Crystallography Analysis of the crystal structures of the two milk-clotting enzymes bovine and camel chymosin has revealed that the better milk-clotting activity towards bovine milk of camel chymosin compared with bovine chymosin is related to variations in their surface charges and their substrate-binding clefts. en ASPARTIC PEPTIDASES; ENZYME ACTIVITY; SUBSTRATE SPECIFICITY; MILK CLOTTING; SURFACE CHARGE; DOMAIN FLEXIBILITY Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central β-sheet that connects the N-terminal and C-­terminal domains. In bovine chymosin the N-terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate-binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk. text/html Camel and bovine chymosin: the relationship between their structures and cheese-making properties text 5 69 2013-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 901 med@iucr.org 913 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd9154 The article by Koval' et al. [(2013). Acta Cryst. D69, 213–226] is corrected. Copyright (c) 2013 International Union of Crystallography urn:issn:0907-4449 Koval', T. Lipovová, P. Podzimek, T. Matoušek, J. Dušková, J. Skálová, T. Štěpánková, A. Hašek, J. Dohnálek, J. 2013-06-01 doi:10.1107/S0907444913003284 International Union of Crystallography A correction is made to the article by Koval' et al. [(2013). Acta Cryst. D69, 213–226]. en PLANT NUCLEASES; CATALYTIC ZINC CLUSTER; GLYCOPROTEINS; ANTITUMOUR ACTIVITY; OLIGOMERIZATION; PHOSPHOLIPASE C-LIKE ACTIVITY; CORRIGENDUM The article by Koval' et al. [(2013). Acta Cryst. D69, 213–226] is corrected. text/html Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis. Corrigendum text 6 69 2013-06-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2013 International Union of Crystallography 0907-4449 addenda and errata 1192 med@iucr.org 1192 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5081 The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-­terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBC). Here, the crystal structures of FlhBC from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhBC structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhBC leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhBC and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhBC molecule. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Meshcheryakov, V.A. Kitao, A. Matsunami, H. Samatey, F.A. 2013-05-01 doi:10.1107/S0907444913002102 International Union of Crystallography Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. en FLHB; BACTERIAL FLAGELLUM; TYPE III SECRETION SYSTEM; SALMONELLA; AQUIFEX The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-­terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBC). Here, the crystal structures of FlhBC from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhBC structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhBC leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhBC and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhBC molecule. text/html Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins text 5 69 2013-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 812 med@iucr.org 820 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dw5040 Plant Deg5 and Deg8 are two members of the HtrA proteases, a family of oligomeric serine endopeptidases that are involved in a variety of protein quality-control processes. These two HtrA proteases are located in the thylakoid lumen and participate in high-light stress responses by collaborating with other chloroplast proteins. Deg5 and Deg8 degrade photodamaged D1 protein of the photosystem II reaction centre, allowing its in situ replacement. Here, the crystal structures of Arabidopsis thaliana Deg5 (S266A) and Deg8 (S292A) are reported at 2.6 and 2.0 Å resolution, respectively. The Deg5 trimer contains two calcium ions in a central channel, suggesting a link between photodamage control and calcium ions in chloroplasts. Previous structures of HtrA proteases have indicated that their regulation usually requires C-terminal PDZ domain(s). Deg5 is unique in that it contains no PDZ domain and the trimeric structure of Deg5 (S266A) reveals a novel catalytic triad conformation. A similar triad conformation is observed in the hexameric structure of the single PDZ-domain-containing Deg8 (S292A). These findings suggest a novel activation mechanism for plant HtrA proteases and provide structural clues to their function in light-stress response. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Sun, W. Gao, F. Fan, H. Shan, X. Sun, R. Liu, L. Gong, W. 2013-05-01 doi:10.1107/S0907444913002023 International Union of Crystallography The crystal structures of Arabidopsis Deg5 and Deg8 have been determined to resolutions of 2.6 and 2.0 Å, respectively, revealing novel structural features of HtrA proteases. en PROTEASE-CHAPERONES; PROTEIN QUALITY CONTROL; PDZ DOMAINS; OLIGOMERIZATION Plant Deg5 and Deg8 are two members of the HtrA proteases, a family of oligomeric serine endopeptidases that are involved in a variety of protein quality-control processes. These two HtrA proteases are located in the thylakoid lumen and participate in high-light stress responses by collaborating with other chloroplast proteins. Deg5 and Deg8 degrade photodamaged D1 protein of the photosystem II reaction centre, allowing its in situ replacement. Here, the crystal structures of Arabidopsis thaliana Deg5 (S266A) and Deg8 (S292A) are reported at 2.6 and 2.0 Å resolution, respectively. The Deg5 trimer contains two calcium ions in a central channel, suggesting a link between photodamage control and calcium ions in chloroplasts. Previous structures of HtrA proteases have indicated that their regulation usually requires C-terminal PDZ domain(s). Deg5 is unique in that it contains no PDZ domain and the trimeric structure of Deg5 (S266A) reveals a novel catalytic triad conformation. A similar triad conformation is observed in the hexameric structure of the single PDZ-domain-containing Deg8 (S292A). These findings suggest a novel activation mechanism for plant HtrA proteases and provide structural clues to their function in light-stress response. text/html The structures of Arabidopsis Deg5 and Deg8 reveal new insights into HtrA proteases text 5 69 2013-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 830 med@iucr.org 837 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5087 The history and the current state of the PDB and EMDB archives is briefly described, as well as some of the challenges that they face. It seems natural that the role of structural biology archives will change from being a pure repository of historic data into becoming an indispensable resource for the wider biomedical community. As part of this transformation, it will be necessary to validate the biomacromolecular structure data and ensure the highest possible quality for the archive holdings, to combine structural data from different spatial scales into a unified resource and to integrate structural data with functional, genetic and taxonomic data as well as other information available in bioinformatics resources. Some recent developments and plans to address these challenges at PDBe are presented. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gutmanas, A. Oldfield, T.J. Patwardhan, A. Sen, S. Velankar, S. Kleywegt, G.J. 2013-05-01 doi:10.1107/S0907444913001157 International Union of Crystallography The integration of structural data on atomistic to cellular scales with genetic, taxonomic and functional information is discussed. The challenges to the PDB and EMDB archives and some pertinent developments at PDBe to address these are discussed. en STRUCTURAL BIOINFORMATICS; INTEGRATIVE STRUCTURAL BIOLOGY; PDBE The history and the current state of the PDB and EMDB archives is briefly described, as well as some of the challenges that they face. It seems natural that the role of structural biology archives will change from being a pure repository of historic data into becoming an indispensable resource for the wider biomedical community. As part of this transformation, it will be necessary to validate the biomacromolecular structure data and ensure the highest possible quality for the archive holdings, to combine structural data from different spatial scales into a unified resource and to integrate structural data with functional, genetic and taxonomic data as well as other information available in bioinformatics resources. Some recent developments and plans to address these challenges at PDBe are presented. text/html The role of structural bioinformatics resources in the era of integrative structural biology text 5 69 2013-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 710 med@iucr.org 721 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5197 Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Langer, G.G. Hazledine, S. Wiegels, T. Carolan, C. Lamzin, V.S. 2013-04-01 doi:10.1107/S0907444913000565 International Union of Crystallography The molecular viewer ArpNavigator allows easy execution of ARP/wARP model-building routines while model-update steps are shown in real time, rendering the whole process transparent to the user. en MODEL BUILDING; ARP/WARP; MOLECULAR GRAPHICS Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user. text/html Visual automated macromolecular model building text 69 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2013-04-01 635 research papers 0907-4449 med@iucr.org 641 1399-0047 http://scripts.iucr.org/cgi-bin/paper?me0493 This is an introduction to four papers based on presentations given at a workshop entitled Integrated Software for Integrative Structural Biology. The use of hybrid techniques, and other trends in structural research, pose new challenges to software developers. A structural biology work bench that meets these needs would provide seamless data transfer between processing steps, and accumulate archival data and metadata without intruding into the scientist's work process. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Morris, C. 2013-05-01 doi:10.1107/S090744491300276X International Union of Crystallography The trends to investigate larger complexes and more transient phenomena pose some challenges to software developers. en SOFTWARE; INTEGRATIVE STRUCTURAL BIOLOGY; STRUCTURAL BIOLOGY This is an introduction to four papers based on presentations given at a workshop entitled Integrated Software for Integrative Structural Biology. The use of hybrid techniques, and other trends in structural research, pose new challenges to software developers. A structural biology work bench that meets these needs would provide seamless data transfer between processing steps, and accumulate archival data and metadata without intruding into the scientist's work process. text/html Towards a structural biology work bench text 5 69 2013-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 681 med@iucr.org 682 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5273 A fast and robust method for determining the parameters for a flat (mask-based) bulk-solvent model and overall scaling in macromolecular crystallographic structure refinement and other related calculations is described. This method uses analytical expressions for the determination of optimal values for various scale factors. The new approach was tested using nearly all entries in the PDB for which experimental structure factors are available. In general, the resulting R factors are improved compared with previously implemented approaches. In addition, the new procedure is two orders of magnitude faster, which has a significant impact on the overall runtime of refinement and other applications. An alternative function is also proposed for scaling the bulk-solvent model and it is shown that it outperforms the conventional exponential function. Similarly, alternative methods are presented for anisotropic scaling and their performance is analyzed. All methods are implemented in the Computational Crystallo­graphy Toolbox (cctbx) and are used in PHENIX programs. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Afonine, P.V. Grosse-Kunstleve, R.W. Adams, P.D. Urzhumtsev, A. 2013-04-01 doi:10.1107/S0907444913000462 International Union of Crystallography A fast analytical method for calculating mask-based bulk-solvent scale factors and overall anisotropic correction factors is introduced. en BULK SOLVENT; SCALING; ANISOTROPY; STRUCTURE REFINEMENT; PHENIX A fast and robust method for determining the parameters for a flat (mask-based) bulk-solvent model and overall scaling in macromolecular crystallographic structure refinement and other related calculations is described. This method uses analytical expressions for the determination of optimal values for various scale factors. The new approach was tested using nearly all entries in the PDB for which experimental structure factors are available. In general, the resulting R factors are improved compared with previously implemented approaches. In addition, the new procedure is two orders of magnitude faster, which has a significant impact on the overall runtime of refinement and other applications. An alternative function is also proposed for scaling the bulk-solvent model and it is shown that it outperforms the conventional exponential function. Similarly, alternative methods are presented for anisotropic scaling and their performance is analyzed. All methods are implemented in the Computational Crystallo­graphy Toolbox (cctbx) and are used in PHENIX programs. text/html Bulk-solvent and overall scaling revisited: faster calculations, improved results text 69 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2013-04-01 625 research papers 0907-4449 med@iucr.org 634 1399-0047 http://scripts.iucr.org/cgi-bin/paper?lv5031 Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the `portal entrance' of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP–SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a `locked' dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the `flexible' and `locked' dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Samygina, V.R. Ochoa-Lizarralde, B. Popov, A.N. Cabo-Bilbao, A. Goni-de-Cerio, F. Molotkovsky, J.G. Patel, D.J. Brown, R.E. Malinina, L. 2013-04-01 doi:10.1107/S0907444913000024 International Union of Crystallography It is shown that dimerization is promoted by glycolipid binding to human GLTP. The importance of dimer flexibility in wild-type protein is manifested by point mutation that `locks' the dimer while diversifying ligand/protein adaptations. en GLYCOLIPID TRANSFER PROTEIN; SELECTIVITY; SULFATIDES; LIPID-MEDIATED HOMODIMERIZATION; GLTP FOLD Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the `portal entrance' of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP–SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a `locked' dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the `flexible' and `locked' dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP. text/html Structural insights into lipid-dependent reversible dimerization of human GLTP text 69 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2013-04-01 603 research papers 0907-4449 med@iucr.org 616 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5267 The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Sayer, C. Isupov, M.N. Westlake, A. Littlechild, J.A. 2013-04-01 doi:10.1107/S0907444912051670 International Union of Crystallography The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. en AMINOTRANSFERASES; TRANSAMINASES; SUBSTRATE SPECIFICITY; INDUSTRIAL BIOCATALYSIS The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. text/html Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity text 69 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2013-04-01 564 research papers 0907-4449 med@iucr.org 576 1399-0047 http://scripts.iucr.org/cgi-bin/paper?me0488 Copyright (c) 2013 International Union of Crystallography urn:issn:0907-4449 Baker, E.N. Dauter, Z. 2013-01-01 doi:10.1107/S0907444912051190 International Union of Crystallography Editorial. en EDITORIAL text/html Celebrating biological crystallography: Acta D twenty years on text 1 69 2013-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2013 International Union of Crystallography 0907-4449 editorial 1 med@iucr.org 1 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5412 Linear motifs normally bind with only medium binding affinity (Kd of ∼0.1–10 µM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein–peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein–peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of `unwanted' crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein–peptide complexes. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gógl, G. Törő, I. Reményi, A. 2013-03-01 doi:10.1107/S0907444912051062 International Union of Crystallography A rational surface-engineering approach led to the crystal structure determination of ERK2–docking peptide complexes. en LINEAR MOTIFS; SURFACE ENGINEERING; ERK2; PROTEIN-PEPTIDE COMPLEXES Linear motifs normally bind with only medium binding affinity (Kd of ∼0.1–10 µM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein–peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein–peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of `unwanted' crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein–peptide complexes. text/html Protein–peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase text 3 69 2013-03-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 short communications 486 med@iucr.org 489 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5217 Fibrous proteins in the amyloid state are found both associated with numerous diseases and in the normal functions of cells. Amyloid fibers contain a repetitive spine, commonly built from a pair of β-sheets whose β-strands run perpendicular to the fiber direction and whose side chains interdigitate, much like the teeth of a zipper. In fiber spines known as homosteric zippers, identical protein segments sharing identical packing environments make the two β-sheets. In previous work based on atomic resolution crystal structures of homosteric zippers derived from a dozen proteins, the symmetries of homosteric zippers were categorized into eight classes. Here, it is shown through a formal derivation that each homosteric zipper class corresponds to a unique set of symmetry groups termed `zipper groups'. Furthermore, the eight previously identified classes do not account for all of the 15 possible zipper groups, which may be categorized into the complete set of ten classes. Because of their foundations in group theory, the 15 zipper groups provide a mathematically rigorous classification for homosteric zippers. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Stroud, J.C. 2013-04-01 doi:10.1107/S0907444912050548 International Union of Crystallography A formal derivation is provided of the 15 symmetry groups (zipper groups) available to the amyloid homosteric zipper. en AMYLOID SPINE; STERIC ZIPPERS; ZIPPER GROUPS; SYMMETRY; GROUP THEORY; AMYLOID FIBERS Fibrous proteins in the amyloid state are found both associated with numerous diseases and in the normal functions of cells. Amyloid fibers contain a repetitive spine, commonly built from a pair of β-sheets whose β-strands run perpendicular to the fiber direction and whose side chains interdigitate, much like the teeth of a zipper. In fiber spines known as homosteric zippers, identical protein segments sharing identical packing environments make the two β-sheets. In previous work based on atomic resolution crystal structures of homosteric zippers derived from a dozen proteins, the symmetries of homosteric zippers were categorized into eight classes. Here, it is shown through a formal derivation that each homosteric zipper class corresponds to a unique set of symmetry groups termed `zipper groups'. Furthermore, the eight previously identified classes do not account for all of the 15 possible zipper groups, which may be categorized into the complete set of ten classes. Because of their foundations in group theory, the 15 zipper groups provide a mathematically rigorous classification for homosteric zippers. text/html The zipper groups of the amyloid state of proteins text 4 69 2013-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 540 med@iucr.org 545 1399-0047 http://scripts.iucr.org/cgi-bin/paper?kw5053 In lactic acid bacteria and other bacteria, carbohydrate uptake is mostly governed by phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). PTS-dependent translocation through the cell membrane is coupled with phosphorylation of the incoming sugar. After translocation through the bacterial membrane, the β-glycosidic bond in 6′-­P-­β-glucoside is cleaved, releasing 6-P-β-glucose and the respective aglycon. This reaction is catalyzed by 6-P-β-glucosidases, which belong to two glycoside hydrolase (GH) families: GH1 and GH4. Here, the high-resolution crystal structures of GH1 6-P-β-glucosidases from Lactobacillus plantarum (LpPbg1) and Streptococcus mutans (SmBgl) and their complexes with ligands are reported. Both enzymes show hydrolytic activity towards 6′-P-β-glucosides. The LpPbg1 structure has been determined in an apo form as well as in a complex with phosphate and a glucose molecule corresponding to the aglycon molecule. The S. mutans homolog contains a sulfate ion in the phosphate-dedicated subcavity. SmBgl was also crystallized in the presence of the reaction product 6-P-β-glucose. For a mutated variant of the S. mutans enzyme (E375Q), the structure of a 6′-P-salicin complex has also been determined. The presence of natural ligands enabled the definition of the structural elements that are responsible for substrate recognition during catalysis. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Michalska, K. Tan, K. Li, H. Hatzos-Skintges, C. Bearden, J. Babnigg, G. Joachimiak, A. 2013-03-01 doi:10.1107/S0907444912049608 International Union of Crystallography The crystal structures of two 6-P-β-glucosidases from the GH1 family were determined in the apo form and in the presence of a 6′-P-salicin substrate, of the reaction product 6-P-β-glucose and of glucose corresponding to the aglycon molecule. The presence of natural ligands enabled the definition of the structural elements responsible for the recognition and hydrolysis of 6′-P-β-glucosides. en 6-P-[BETA]-GLUCOSIDASES; GLYCOSIDE HYDROLASES; GH1; CELLOBIOSE; GENTIOBIOSE; SALICIN In lactic acid bacteria and other bacteria, carbohydrate uptake is mostly governed by phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). PTS-dependent translocation through the cell membrane is coupled with phosphorylation of the incoming sugar. After translocation through the bacterial membrane, the β-glycosidic bond in 6′-­P-­β-glucoside is cleaved, releasing 6-P-β-glucose and the respective aglycon. This reaction is catalyzed by 6-P-β-glucosidases, which belong to two glycoside hydrolase (GH) families: GH1 and GH4. Here, the high-resolution crystal structures of GH1 6-P-β-glucosidases from Lactobacillus plantarum (LpPbg1) and Streptococcus mutans (SmBgl) and their complexes with ligands are reported. Both enzymes show hydrolytic activity towards 6′-P-β-glucosides. The LpPbg1 structure has been determined in an apo form as well as in a complex with phosphate and a glucose molecule corresponding to the aglycon molecule. The S. mutans homolog contains a sulfate ion in the phosphate-dedicated subcavity. SmBgl was also crystallized in the presence of the reaction product 6-P-β-glucose. For a mutated variant of the S. mutans enzyme (E375Q), the structure of a 6′-P-salicin complex has also been determined. The presence of natural ligands enabled the definition of the structural elements that are responsible for substrate recognition during catalysis. text/html GH1-family 6-P-β-glucosidases from human microbiome lactic acid bacteria text 3 69 2013-03-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 451 med@iucr.org 463 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5198 The transpeptidase LtdMt2 catalyzes the formation of the (3–3) cross-links characteristic of the peptidoglycan layer in the Mycobacterium tuberculosis cell wall. Bioinformatics analysis suggests that the extramembrane part of the enzyme consists of three domains: two smaller domains (denoted as A and B domains) and a transpeptidase domain (the C domain) at the C-­terminus. The crystal structures of two fragments comprising the AB domains and the BC domains have been determined. The structure of the BC module, which was determined to 1.86 Å resolution using Se-SAD phasing, consists of the B domain with an immunoglobulin-related fold and the catalytic domain belonging to the ErfK/YbiS/YbnG fold family. The structure of the AB-domain fragment, which was solved by molecular replacement to 1.45 Å resolution, reveals that despite a lack of overall sequence identity the A domain is structurally very similar to the B domain. Combining the structures of the two fragments provides a view of the complete three-domain extramembrane part of LdtMt2 and shows that the protein extends at least 80–100 Å from the plasma membrane into the peptidoglycan layer and thus defines the maximal distance at which cross-links are formed by this enzyme. The LdtMt-related transpeptidases contain one or two immunoglobulin domains, which suggests that these might serve as extender units to position the catalytic domain at an appropriate distance from the membrane in the peptidoglycan layer. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Böth, D. Steiner, E.M. Stadler, D. Lindqvist, Y. Schnell, R. Schneider, G. 2013-03-01 doi:10.1107/S0907444912049268 International Union of Crystallography The crystal structures of two fragments of the l,d-transpeptidase from M. tuberculosis have been determined at 1.45 and 1.86 Å resolution. The extramembrane part of this enzyme consists of three domains: two domains related to the immunoglobulin fold and a catalytic domain belonging to the ErfK/YbiS/YhnG family at the C-terminus. en MYCOBACTERIUM TUBERCULOSIS; CELL WALL; PEPTIDOGLYCANS; TRANSPEPTIDASES; ANTIBIOTICS The transpeptidase LtdMt2 catalyzes the formation of the (3–3) cross-links characteristic of the peptidoglycan layer in the Mycobacterium tuberculosis cell wall. Bioinformatics analysis suggests that the extramembrane part of the enzyme consists of three domains: two smaller domains (denoted as A and B domains) and a transpeptidase domain (the C domain) at the C-­terminus. The crystal structures of two fragments comprising the AB domains and the BC domains have been determined. The structure of the BC module, which was determined to 1.86 Å resolution using Se-SAD phasing, consists of the B domain with an immunoglobulin-related fold and the catalytic domain belonging to the ErfK/YbiS/YbnG fold family. The structure of the AB-domain fragment, which was solved by molecular replacement to 1.45 Å resolution, reveals that despite a lack of overall sequence identity the A domain is structurally very similar to the B domain. Combining the structures of the two fragments provides a view of the complete three-domain extramembrane part of LdtMt2 and shows that the protein extends at least 80–100 Å from the plasma membrane into the peptidoglycan layer and thus defines the maximal distance at which cross-links are formed by this enzyme. The LdtMt-related transpeptidases contain one or two immunoglobulin domains, which suggests that these might serve as extender units to position the catalytic domain at an appropriate distance from the membrane in the peptidoglycan layer. text/html Structure of LdtMt2, an l,d-transpeptidase from Mycobacterium tuberculosis text 3 69 2013-03-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 432 med@iucr.org 441 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5223 Difficulty in the treatment of tuberculosis and growing drug resistance in Mycobacterium tuberculosis (Mtb) are a global health issue. Carbapenems inactivate l,d-transpeptidases; meropenem, when administered with clavulanate, showed in vivo activity against extensively drug-resistant Mtb strains. LdtMt2 (Rv2518c), one of two functional l,d-transpeptidases in Mtb, is predominantly expressed over LdtMt1 (Rv0116c). Here, the crystal structure of N-terminally truncated LdtMt2 (residues Leu131–Ala408) is reported in both ligand-free and meropenem-bound forms. The structure of meropenem-inhibited LdtMt2 provides a detailed structural view of the interactions between a carbapenem drug and Mtb l,d-transpeptidase. The structures revealed that the catalytic l,d-­transpeptidase domain of LdtMt2 is preceded by a bacterial immunogloblin-like Big_5 domain and is followed by an extended C-terminal tail that interacts with both domains. Furthermore, it is shown using mass analyses that meropenem acts as a suicide inhibitor of LdtMt2. Upon acylation of the catalytic Cys354 by meropenem, the `active-site lid' undergoes a large conformational change to partially cover the active site so that the bound meropenem is accessible to the bulk solvent via three narrow paths. This work will facilitate structure-guided discovery of l,d-transpeptidase inhibitors as novel antituberculosis drugs against drug-resistant Mtb. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kim, H.S. Kim, J. Im, H.N. Yoon, J.Y. An, D.R. Yoon, H.J. Kim, J.Y. Min, H.K. Kim, S.-J. Lee, J.Y. Han, B.W. Suh, S.W. 2013-03-01 doi:10.1107/S0907444912048998 International Union of Crystallography The crystal structure of M. tuberculosis l,d-transpeptidase (LdtMt2; Rv2518c) has been determined in both ligand-free and meropenem-bound forms. The detailed view of the interactions between meropenem and LdtMt2 will be useful in structure-guided discovery of new antituberculosis drugs. en RV2518C; MT2594; LDTMT2; L,D-TRANSPEPTIDASES; MYCOBACTERIUM TUBERCULOSIS; MEROPENEM; CARBAPENEM; PEPTIDOGLYCANS; ANTITUBERCULOSIS DRUG DISCOVERY Difficulty in the treatment of tuberculosis and growing drug resistance in Mycobacterium tuberculosis (Mtb) are a global health issue. Carbapenems inactivate l,d-transpeptidases; meropenem, when administered with clavulanate, showed in vivo activity against extensively drug-resistant Mtb strains. LdtMt2 (Rv2518c), one of two functional l,d-transpeptidases in Mtb, is predominantly expressed over LdtMt1 (Rv0116c). Here, the crystal structure of N-terminally truncated LdtMt2 (residues Leu131–Ala408) is reported in both ligand-free and meropenem-bound forms. The structure of meropenem-inhibited LdtMt2 provides a detailed structural view of the interactions between a carbapenem drug and Mtb l,d-transpeptidase. The structures revealed that the catalytic l,d-­transpeptidase domain of LdtMt2 is preceded by a bacterial immunogloblin-like Big_5 domain and is followed by an extended C-terminal tail that interacts with both domains. Furthermore, it is shown using mass analyses that meropenem acts as a suicide inhibitor of LdtMt2. Upon acylation of the catalytic Cys354 by meropenem, the `active-site lid' undergoes a large conformational change to partially cover the active site so that the bound meropenem is accessible to the bulk solvent via three narrow paths. This work will facilitate structure-guided discovery of l,d-transpeptidase inhibitors as novel antituberculosis drugs against drug-resistant Mtb. text/html Structural basis for the inhibition of Mycobacterium tuberculosis l,d-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains text 3 69 2013-03-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 420 med@iucr.org 431 1399-0047 http://scripts.iucr.org/cgi-bin/paper?me0485 Copyright (c) 2013 International Union of Crystallography urn:issn:0907-4449 Dauter, Z. Weiss, M.S. Einspahr, H. Baker, E.N. 2013-02-01 doi:10.1107/S0907444913000255 International Union of Crystallography Editorial. en EDITORIAL text/html Expectation bias and information content text 2 69 2013-02-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2013 International Union of Crystallography 0907-4449 editorial 141 med@iucr.org 141 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5185 Heavy-atom clusters (HA clusters) containing a large number of specifically arranged electron-dense scatterers are especially useful for experimental phase determination of large complex structures, weakly diffracting crystals or structures with large unit cells. Often, the determination of the exact orientation of the HA cluster and hence of the individual heavy-atom positions proves to be the critical step in successful phasing and subsequent structure solution. Here, it is demonstrated that molecular replacement (MR) with either anomalous or isomorphous differences is a useful strategy for the correct placement of HA cluster compounds. The polyoxometallate cluster hexasodium α-metatungstate (HMT) was applied in phasing the structure of death receptor 6. Even though the HA cluster is bound in alternate partially occupied orientations and is located at a special position, its correct localization and orientation could be determined at resolutions as low as 4.9 Å. The broad applicability of this approach was demonstrated for five different derivative crystals that included the compounds tantalum tetradeca­bromide and trisodium phosphotungstate in addition to HMT. The correct placement of the HA cluster depends on the length of the intramolecular vectors chosen for MR, such that both a larger cluster size and the optimal choice of the wavelength used for anomalous data collection strongly affect the outcome. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Dahms, S.O. Kuester, M. Streb, C. Roth, C. Sträter, N. Than, M.E. 2013-02-01 doi:10.1107/S0907444912046008 International Union of Crystallography A new approach is presented that allows the efficient localization and orientation of heavy-atom cluster compounds used in experimental phasing by a molecular replacement procedure. This permits the calculation of meaningful phases up to the highest resolution of the diffraction data. en EXPERIMENTAL PHASING; HEAVY-METAL CLUSTER; HEXASODIUM [ALPHA]-METATUNGSTATE; MOLECULAR REPLACEMENT; DEATH RECEPTOR 6 Heavy-atom clusters (HA clusters) containing a large number of specifically arranged electron-dense scatterers are especially useful for experimental phase determination of large complex structures, weakly diffracting crystals or structures with large unit cells. Often, the determination of the exact orientation of the HA cluster and hence of the individual heavy-atom positions proves to be the critical step in successful phasing and subsequent structure solution. Here, it is demonstrated that molecular replacement (MR) with either anomalous or isomorphous differences is a useful strategy for the correct placement of HA cluster compounds. The polyoxometallate cluster hexasodium α-metatungstate (HMT) was applied in phasing the structure of death receptor 6. Even though the HA cluster is bound in alternate partially occupied orientations and is located at a special position, its correct localization and orientation could be determined at resolutions as low as 4.9 Å. The broad applicability of this approach was demonstrated for five different derivative crystals that included the compounds tantalum tetradeca­bromide and trisodium phosphotungstate in addition to HMT. The correct placement of the HA cluster depends on the length of the intramolecular vectors chosen for MR, such that both a larger cluster size and the optimal choice of the wavelength used for anomalous data collection strongly affect the outcome. text/html Localization and orientation of heavy-atom cluster compounds in protein crystals using molecular replacement text 2 69 2013-02-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 284 med@iucr.org 297 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5268 In the case of translational noncrystallographic symmetry (tNCS), two or more copies of a component in the asymmetric unit of the crystal are present in a similar orientation. This causes systematic modulations of the reflection intensities in the diffraction pattern, leading to problems with structure determination and refinement methods that assume, either implicitly or explicitly, that the distribution of intensities is a function only of resolution. To characterize the statistical effects of tNCS accurately, it is necessary to determine the translation relating the copies, any small rotational differences in their orientations, and the size of random coordinate differences caused by conformational differences. An algorithm to estimate these parameters and refine their values against a likelihood function is presented, and it is shown that by accounting for the statistical effects of tNCS it is possible to unmask the competing statistical effects of twinning and tNCS and to more robustly assess the crystal for the presence of twinning. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Read, R.J. Adams, P.D. McCoy, A.J. 2013-02-01 doi:10.1107/S0907444912045374 International Union of Crystallography The statistical effects of translational noncrystallographic symmetry can be characterized by maximizing parameters describing the noncrystallographic symmetry in a likelihood function, thereby unmasking the competing statistical effects of twinning. en TRANSLATIONAL NONCRYSTALLOGRAPHIC SYMMETRY; INTENSITY STATISTICS; TWINNING; MAXIMUM LIKELIHOOD In the case of translational noncrystallographic symmetry (tNCS), two or more copies of a component in the asymmetric unit of the crystal are present in a similar orientation. This causes systematic modulations of the reflection intensities in the diffraction pattern, leading to problems with structure determination and refinement methods that assume, either implicitly or explicitly, that the distribution of intensities is a function only of resolution. To characterize the statistical effects of tNCS accurately, it is necessary to determine the translation relating the copies, any small rotational differences in their orientations, and the size of random coordinate differences caused by conformational differences. An algorithm to estimate these parameters and refine their values against a likelihood function is presented, and it is shown that by accounting for the statistical effects of tNCS it is possible to unmask the competing statistical effects of twinning and tNCS and to more robustly assess the crystal for the presence of twinning. text/html Intensity statistics in the presence of translational noncrystallographic symmetry text 2 69 2013-02-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 176 med@iucr.org 183 1399-0047 http://scripts.iucr.org/cgi-bin/paper?me0482 Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Baker, E.N. Dauter, Z. 2012-11-01 doi:10.1107/S0907444912043107 International Union of Crystallography Editorial. en EDITORIAL text/html Nobel Prize for Chemistry 2012: GPCRs seen through the crystal ball text 11 68 2012-11-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 editorial 1439 med@iucr.org 1440 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dw5029 Many pathogenic bacteria that infect humans, animals and plants rely on a quorum-sensing (QS) system to produce virulence factors. N-Acyl homoserine lactones (AHLs) are the best-characterized cell–cell communication signals in QS. The concentration of AHL plays a key role in regulating the virulence-gene expression and essential biological functions of pathogenic bacteria. N-Acyl homoserine lactonases (AHL-lactonases) have important functions in decreasing pathogenicity by degrading AHLs. Here, structures of the AHL-lactonase from Ochrobactrum sp. (AidH) in complex with N-­hexanoyl homoserine lactone, N-hexanoyl homoserine and N-­butanoyl homoserine are reported. The high-resolution structures together with biochemical analyses reveal convincing details of AHL degradation. No metal ion is bound in the active site, which is different from other AHL-lactonases, which have a dual Lewis acid catalysis mechanism. AidH contains a substrate-binding tunnel between the core domain and the cap domain. The conformation of the tunnel entrance varies with the AHL acyl-chain length, which contributes to the binding promiscuity of AHL molecules in the active site. It also supports the biochemical result that AidH is a broad catalytic spectrum AHL-lactonase. Taken together, the present results reveal the catalytic mechanism of the metal-independent AHL-lactonase, which is a typical acid–base covalent catalysis. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gao, A. Mei, G. Liu, S. Wang, P. Tang, Q. Liu, Y. Wen, H. An, X. Zhang, L. Yan, X. Liang, D. 2013-01-01 doi:10.1107/S0907444912042369 International Union of Crystallography Crystal structures of the AHL-lactonase AidH in complex with substrate and product are reported at high resolution and a catalytic mechanism is proposed for the metal-independent AHL-lactonase. en QUORUM SENSING; LACTONASES; [ALPHA]/[BETA]-HYDROLASES; ACID-BASE CATALYSIS; COVALENT CATALYSIS Many pathogenic bacteria that infect humans, animals and plants rely on a quorum-sensing (QS) system to produce virulence factors. N-Acyl homoserine lactones (AHLs) are the best-characterized cell–cell communication signals in QS. The concentration of AHL plays a key role in regulating the virulence-gene expression and essential biological functions of pathogenic bacteria. N-Acyl homoserine lactonases (AHL-lactonases) have important functions in decreasing pathogenicity by degrading AHLs. Here, structures of the AHL-lactonase from Ochrobactrum sp. (AidH) in complex with N-­hexanoyl homoserine lactone, N-hexanoyl homoserine and N-­butanoyl homoserine are reported. The high-resolution structures together with biochemical analyses reveal convincing details of AHL degradation. No metal ion is bound in the active site, which is different from other AHL-lactonases, which have a dual Lewis acid catalysis mechanism. AidH contains a substrate-binding tunnel between the core domain and the cap domain. The conformation of the tunnel entrance varies with the AHL acyl-chain length, which contributes to the binding promiscuity of AHL molecules in the active site. It also supports the biochemical result that AidH is a broad catalytic spectrum AHL-lactonase. Taken together, the present results reveal the catalytic mechanism of the metal-independent AHL-lactonase, which is a typical acid–base covalent catalysis. text/html High-resolution structures of AidH complexes provide insights into a novel catalytic mechanism for N-acyl homoserine lactonase text 1 69 2013-01-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 82 med@iucr.org 91 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dw5030 Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1–2 and a deletion near site Ca3. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Ruf, A. Stihle, M. Benz, J. Schmidt, M. Sobek, H. 2013-01-01 doi:10.1107/S0907444912041169 International Union of Crystallography The crystal structure of the metalloprotease Gentlyase is described and compared with the structures of other related thermolysin-like proteases. en THERMOLYSIN; NEUTRAL PROTEASES; CALCIUM BINDING; GENTLYASE Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1–2 and a deletion near site Ca3. text/html Structure of Gentlyase, the neutral metalloprotease of Paenibacillus polymyxa text 1 69 2013-01-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 24 med@iucr.org 31 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5261 When embarking upon X-ray diffraction data collection from a potentially novel macromolecular crystal form, it can be useful to ascertain whether the measured data reflect a crystal form that is already recorded in the Protein Data Bank and, if so, whether it is part of a large family of related structures. Providing such information to crystallographers conveniently and quickly, as soon as the first images have been recorded and the unit cell characterized at an X-ray beamline, has the potential to save time and effort as well as pointing to possible search models for molecular replacement. Given an input unit cell, and optionally a space group, Nearest-cell rapidly scans the Protein Data Bank and retrieves near-matches. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Ramraj, V. Evans, G. Diprose, J.M. Esnouf, R.M. 2012-12-01 doi:10.1107/S0907444912040590 International Union of Crystallography A fast and easy tool to locate unit-cell matches in the PDB is described. en NEAREST-CELL; CRYSTAL MATCHES; PROTEIN DATA BANK When embarking upon X-ray diffraction data collection from a potentially novel macromolecular crystal form, it can be useful to ascertain whether the measured data reflect a crystal form that is already recorded in the Protein Data Bank and, if so, whether it is part of a large family of related structures. Providing such information to crystallographers conveniently and quickly, as soon as the first images have been recorded and the unit cell characterized at an X-ray beamline, has the potential to save time and effort as well as pointing to possible search models for molecular replacement. Given an input unit cell, and optionally a space group, Nearest-cell rapidly scans the Protein Data Bank and retrieves near-matches. text/html Nearest-cell: a fast and easy tool for locating crystal matches in the PDB text 68 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-12-01 1697 short communications 0907-4449 med@iucr.org 1700 1399-0047 http://scripts.iucr.org/cgi-bin/paper?cb5015 Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the α(1–2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C—H⋯O hydrogen bonds facilitated by the α(1–2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O3 and O4 atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O1 atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Hoque, M.M. Suzuki, K. Tsunoda, M. Jiang, J. Zhang, F. Takahashi, A. Ohbayashi, N. Zhang, X. Tanaka, H. Ōmura, S. Takénaka, A. 2012-12-01 doi:10.1107/S0907444912040498 International Union of Crystallography X-ray analysis of anti-HIV actinohivin in complex with the target α(1-2)mannobiose moiety of high-mannose type glycans attached to HIV-1 gp120 reveals that the three rotamers generated with 120 rotations around the molecular pseudo-rotation axis are packed randomly in the unit cell according to the P212121 symmetry to exhibit an apparent space group P213 as the statistical structure. However, the high-resolution X-ray structure shows the detailed interaction geometry for specific binding. en ANTI-HIV LECTINS; ACTINOHIVIN; HIGH-MANNOSE-TYPE GLYCAN Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the α(1–2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C—H⋯O hydrogen bonds facilitated by the α(1–2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O3 and O4 atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O1 atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120. text/html Structural insights into the specific anti-HIV property of actinohivin: structure of its complex with the α(1–2)mannobiose moiety of gp120 text 68 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-12-01 1671 research papers 0907-4449 med@iucr.org 1679 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5262 SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Bhaskaran, S.S. Stebbins, C.E. 2012-12-01 doi:10.1107/S0907444912039042 International Union of Crystallography The C-terminal domain of the Salmonella virulence factor SseI is structurally similar to the cysteine protease superfamily and contains the conserved catalytic triad characteristic of members of this family. en SALMONELLA; TYPE III SECRETION; SPI-2; BACTERIAL PATHOGENESIS; CYSTEINE PROTEASES; SSEI; SRFH SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells. text/html Structure of the catalytic domain of the Salmonella virulence factor SseI text 68 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-12-01 1613 research papers 0907-4449 med@iucr.org 1621 1399-0047 http://scripts.iucr.org/cgi-bin/paper?xb9052 Additional references are published for the article by Dong et al. [(2012), Acta Cryst. D68, 1134–1139]. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Dong, C. Yang, X. Hou, H.-F. Shen, Y.-Q. Dong, Y.-H. 2012-10-01 doi:10.1107/S0907444912037869 International Union of Crystallography An addendum to the article by Dong et al. [(2012), Acta Cryst. D68, 1134–1139]. en BAM COMPLEX; OUTER MEMBRANE PROTEINS; LIPOPLOYSACCHARIDES; POTRA DOMAINS; ADDENDUM Additional references are published for the article by Dong et al. [(2012), Acta Cryst. D68, 1134–1139]. text/html Structure of Escherichia coli BamB and its interaction with POTRA domains of BamA. Addendum text 10 68 2012-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 addenda and errata 1437 med@iucr.org 1437 1399-0047 http://scripts.iucr.org/cgi-bin/paper?rr5022 The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Liebschner, D. Dauter, M. Rosenbaum, G. Dauter, Z. 2012-10-01 doi:10.1107/S0907444912034658 International Union of Crystallography A repetitive measurement of the same diffraction image allows to judge the performance of a data collection facility. en DIFFRACTION DATA PRECISION; SIGNAL-TO-NOISE RATIO; MEASUREMENT UNCERTAINTY; BEAMLINE PERFORMANCE The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline. text/html How good can our beamlines be? text 68 10 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-10-01 1430 research papers 0907-4449 med@iucr.org 1436 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5408 WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Hagelueken, G. Huang, H. Harlos, K. Clarke, B. Whitfield, C. Naismith, J.H. 2012-10-01 doi:10.1107/S0907444912029599 International Union of Crystallography The optimization of WbdD crystals using a novel dehydration protocol and experimental phasing at 3.5 Å resolution by cross-crystal averaging followed by molecular replacement of electron density into a non-isomorphous 3.0 Å resolution native data set are reported. en WBDD; CRYSTAL DEHYDRATION WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations. text/html Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase from Escherichia coli O9a text 10 68 2012-10-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 1371 med@iucr.org 1379 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dw5015 Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-­ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Halavaty, A.S. Kim, Y. Minasov, G. Shuvalova, L. Dubrovska, I. Winsor, J. Zhou, M. Onopriyenko, O. Skarina, T. Papazisi, L. Kwon, K. Peterson, S.N. Joachimiak, A. Savchenko, A. Anderson, W.F. 2012-10-01 doi:10.1107/S0907444912029101 International Union of Crystallography The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. en ACYL-CARRIER-PROTEIN SYNTHASE; ACYL CARRIER PROTEIN; TYPE II FATTY-ACID SYNTHESIS; INHIBITION; 3',5'-ADENOSINE DIPHOSPHATE; COENZYME A Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-­ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP. text/html Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria text 10 68 2012-10-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 1359 med@iucr.org 1370 1399-0047 http://scripts.iucr.org/cgi-bin/paper?yt5043 The human kinesin Eg5 is responsible for bipolar spindle formation during early mitosis. Inhibition of Eg5 triggers the formation of monoastral spindles, leading to mitotic arrest that eventually causes apoptosis. There is increasing evidence that Eg5 constitutes a potential drug target for the development of cancer chemotherapeutics. The most advanced Eg5-targeting agent is ispinesib, which exhibits potent antitumour activity and is currently in multiple phase II clinical trials. In this study, the crystal structure of the Eg5 motor domain in complex with ispinesib, supported by kinetic and thermodynamic binding data, is reported. Ispinesib occupies the same induced-fit pocket in Eg5 as other allosteric inhibitors, making extensive hydrophobic interactions with the protein. The data for the Eg5–ADP–ispinesib complex suffered from pseudo-mero­hedral twinning and revealed translational noncrystallographic symmetry, leading to challenges in data processing, space-group assignment and structure solution as well as in refinement. These complications may explain the lack of available structural information for this important agent and its analogues. The present structure represents the best interpretation of these data based on extensive data-reduction, structure-solution and refinement trials. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Talapatra, S.K. Schüttelkopf, A.W. Kozielski, F. 2012-10-01 doi:10.1107/S0907444912027965 International Union of Crystallography The complex between the motor protein Eg5 and the phase II clinical candidate ispinesib provides insights into the mechanism of action of this important class of inhibitors. en EG5; KINESINS; ISPINESIB The human kinesin Eg5 is responsible for bipolar spindle formation during early mitosis. Inhibition of Eg5 triggers the formation of monoastral spindles, leading to mitotic arrest that eventually causes apoptosis. There is increasing evidence that Eg5 constitutes a potential drug target for the development of cancer chemotherapeutics. The most advanced Eg5-targeting agent is ispinesib, which exhibits potent antitumour activity and is currently in multiple phase II clinical trials. In this study, the crystal structure of the Eg5 motor domain in complex with ispinesib, supported by kinetic and thermodynamic binding data, is reported. Ispinesib occupies the same induced-fit pocket in Eg5 as other allosteric inhibitors, making extensive hydrophobic interactions with the protein. The data for the Eg5–ADP–ispinesib complex suffered from pseudo-mero­hedral twinning and revealed translational noncrystallographic symmetry, leading to challenges in data processing, space-group assignment and structure solution as well as in refinement. These complications may explain the lack of available structural information for this important agent and its analogues. The present structure represents the best interpretation of these data based on extensive data-reduction, structure-solution and refinement trials. text/html The structure of the ternary Eg5–ADP–ispinesib complex text 10 68 2012-10-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 1311 med@iucr.org 1319 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5183 Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Cha, S.-S. An, Y.J. Jeong, C.-S. Kim, M.-K. Lee, S.-G. Lee, K.-H. Oh, B.-H. 2012-09-01 doi:10.1107/S0907444912024420 International Union of Crystallography The surface of proteins can be charged with zinc ions and the anomalous signals from these zinc ions can be used for structure determination of proteins. en ZINC ANOMALOUS SCATTERING; PHASING; ZN DERIVATIZATION Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination. text/html Experimental phasing using zinc anomalous scattering text 9 68 2012-09-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 short communications 1253 med@iucr.org 1258 1399-0047 http://scripts.iucr.org/cgi-bin/paper?en9495 A correction is published to Table 2 in the article by Cousido-Siah et al. [(2012), Acta Cryst. D68, 1098–1107]. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Cousido-Siah, A. Ayoub, D. Berberián, G. Bollo, M. Van Dorsselaer, A. Debaene, F. DiPolo, R. Petrova, T. Schulze-Briese, C. Olieric, V. Esteves, A. Mitschler, A. Sanglier-Cianférani, S. Beaugé, L. Podjarny, A. 2012-10-01 doi:10.1107/S090744491203898X International Union of Crystallography An erratum to the article by Cousido-Siah et al. [(2012), Acta Cryst. D68, 1098–1107]. en FABP PROTEINS; PALMITIC ACID; SQUID NERVE NA+/CA2+ EXCHANGER REGULATION; ERRATUM A correction is published to Table 2 in the article by Cousido-Siah et al. [(2012), Acta Cryst. D68, 1098–1107]. text/html Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+. Erratum text 10 68 2012-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 addenda and errata 1438 med@iucr.org 1438 1399-0047 http://scripts.iucr.org/cgi-bin/paper?cb5008 Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Dorfmueller, H.C. Fang, W. Rao, F.V. Blair, D.E. Attrill, H. van Aalten, D.M.F. 2012-08-01 doi:10.1107/S0907444912019592 International Union of Crystallography Glucosamine-6-phosphate N-acetyltransferase is an essential enzyme of the eukaryotic UDP-GlcNAc biosynthetic pathway. A crystal structure at 1.55 Å resolution revealed a highly unusual covalent product complex and biochemical studies investigated the function of a fully conserved active-site cysteine. en CARBOHYDRATES; GLYCOBIOLOGY; CAENORHABDITIS ELEGANS; GLUCOSAMINE-6-PHOSPHATE N-ACETYLTRANSFERASE; COENZYME A ADDUCT; MECHANISM Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site. text/html Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1 text 68 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-08-01 1019 research papers 0907-4449 med@iucr.org 1029 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gm5021 The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal re­orientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Brockhauser, S. Svensson, O. Bowler, M.W. Nanao, M. Gordon, E. Leal, R.M.F. Popov, A. Gerring, M. McCarthy, A.A. Gotz, A. 2012-08-01 doi:10.1107/S090744491201863X International Union of Crystallography A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines. This tool provides the possibility of automating complex experiments without the need for expertise in instrumentation control and programming, but rather by accessing defined beamline services. en WORKFLOWS; AUTOMATION; DATA PROCESSING; MACROMOLECULAR CRYSTALLOGRAPHY; EXPERIMENTAL PROTOCOLS; CHARACTERIZATION; REORIENTATION; RADIATION DAMAGE The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal re­orientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE. text/html The use of workflows in the design and implementation of complex experiments in macromolecular crystallography text 68 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-08-01 975 research papers 0907-4449 med@iucr.org 984 1399-0047 http://scripts.iucr.org/cgi-bin/paper?rr5014 RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Keating, K.S. Pyle, A.M. 2012-08-01 doi:10.1107/S0907444912018549 International Union of Crystallography RCrane is a new tool for the partially automated building of RNA crystallographic models into electron-density maps of low or intermediate resolution. This tool helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. en RCRANE; RNA MODEL BUILDING RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems. text/html RCrane: semi-automated RNA model building text 68 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-08-01 985 research papers 0907-4449 med@iucr.org 995 1399-0047 http://scripts.iucr.org/cgi-bin/paper?me0473 Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Baker, E.N. Dauter, Z. 2012-06-01 doi:10.1107/S0907444912021105 International Union of Crystallography Editorial. en EDITORIAL text/html Small angle scattering – moving forward text 6 68 2012-06-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 editorial 619 med@iucr.org 619 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be9113 Residue 235 given in the article by Prabu et al. [(2008). Acta Cryst. D64, 1146–1157] is corrected. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Prabu, J.R. Manjunath, G.P. Chandra, N.R. Muniyappa, K. Vijayan, M. 2012-07-01 doi:10.1107/S0907444912018082 International Union of Crystallography A correction is made to the article by Prabu et al. [(2008). Acta Cryst. D64, 1146–1157]. en RECA; MUTATIONS; ALLOSTERIC TRANSFORMATIONS; CORRIGENDUM Residue 235 given in the article by Prabu et al. [(2008). Acta Cryst. D64, 1146–1157] is corrected. text/html Functionally important movements in RecA molecules and filaments: studies involving mutation and environmental changes. Corrigendum text 7 68 2012-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 addenda and errata 871 med@iucr.org 871 1399-0047 http://scripts.iucr.org/cgi-bin/paper?rr5017 All-atom models are essential for many applications in molecular modeling and computational chemistry. Non­bonded atomic contacts much closer than the sum of the van der Waals radii of the two atoms (clashes) are commonly observed in such models derived from protein crystal structures. A set of 94 recently deposited protein structures in the resolution range 1.5–2.8 Å were analyzed for clashes by the addition of all H atoms to the models followed by optimization and energy minimization of the positions of just these H atoms. The results were compared with the same set of structures after automated all-atom refinement with PrimeX and with nonbonded contacts in protein crystal structures at a resolution equal to or better than 0.9 Å. The additional PrimeX refinement produced structures with reasonable summary geometric statistics and similar Rfree values to the original structures. The frequency of clashes at less than 0.8 times the sum of van der Waals radii was reduced over fourfold compared with that found in the original structures, to a level approaching that found in the ultrahigh-resolution structures. Moreover, severe clashes at less than or equal to 0.7 times the sum of atomic radii were reduced 15-­fold. All-atom refinement with PrimeX produced improved crystal structure models with respect to nonbonded contacts and yielded changes in structural details that dramatically impacted on the interpretation of some protein–ligand interactions. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Bell, J.A. Ho, K.L. Farid, R. 2012-08-01 doi:10.1107/S0907444912017453 International Union of Crystallography All-atom models derived from moderate-resolution protein crystal structures contain a high frequency of close nonbonded contacts, independent of the major refinement program used for structure determination. All-atom refinement with PrimeX corrects many of these problematic interactions, producing models that are better suited for use in computational chemistry and related applications. en H ATOMS; VAN DER WAALS RADII; RESTRAINTS; NONBONDED CONTACTS; CLASHES; MOLECULAR GEOMETRY; MODEL QUALITY; FORCE FIELDS; REFINEMENT; RIDING H ATOMS; ELECTROSTATICS; HYDROGEN BONDS All-atom models are essential for many applications in molecular modeling and computational chemistry. Non­bonded atomic contacts much closer than the sum of the van der Waals radii of the two atoms (clashes) are commonly observed in such models derived from protein crystal structures. A set of 94 recently deposited protein structures in the resolution range 1.5–2.8 Å were analyzed for clashes by the addition of all H atoms to the models followed by optimization and energy minimization of the positions of just these H atoms. The results were compared with the same set of structures after automated all-atom refinement with PrimeX and with nonbonded contacts in protein crystal structures at a resolution equal to or better than 0.9 Å. The additional PrimeX refinement produced structures with reasonable summary geometric statistics and similar Rfree values to the original structures. The frequency of clashes at less than 0.8 times the sum of van der Waals radii was reduced over fourfold compared with that found in the original structures, to a level approaching that found in the ultrahigh-resolution structures. Moreover, severe clashes at less than or equal to 0.7 times the sum of atomic radii were reduced 15-­fold. All-atom refinement with PrimeX produced improved crystal structure models with respect to nonbonded contacts and yielded changes in structural details that dramatically impacted on the interpretation of some protein–ligand interactions. text/html Significant reduction in errors associated with nonbonded contacts in protein crystal structures: automated all-atom refinement with PrimeX text 8 68 2012-08-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 935 med@iucr.org 952 1399-0047 http://scripts.iucr.org/cgi-bin/paper?nj5116 Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-­methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Villaseñor, A.G. Wong, A. Shao, A. Garg, A. Donohue, T.J. Kuglstatter, A. Harris, S.F. 2012-08-01 doi:10.1107/S0907444912016617 International Union of Crystallography Acoustic droplet ejection achieves precise, tipless, non-invasive transfer of diverse aqueous solutions, enabling nanolitre-scale crystallization trials. The rapid and scalable technique demonstrated successful crystal growth with diverse targets in drop volumes as small as 20 nl. en ACOUSTIC LIQUID TRANSFER; NANOLITRE-SCALE CRYSTALLIZATION Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-­methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format. text/html Nanolitre-scale crystallization using acoustic liquid-transfer technology text 68 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-08-01 893 research papers 0907-4449 med@iucr.org 900 1399-0047 http://scripts.iucr.org/cgi-bin/paper?kw5044 An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Å radius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Å r.m.s.d. representing 65–100% of the structure. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Terwilliger, T.C. Read, R.J. Adams, P.D. Brunger, A.T. Afonine, P.V. Grosse-Kunstleve, R.W. Hung, L.-W. 2012-07-01 doi:10.1107/S0907444912015636 International Union of Crystallography A density-based procedure is described for improving a homology model that is locally accurate but differs globally. The model is deformed to match the map and refined, yielding an improved starting point for density modification and further model-building. en MOLECULAR REPLACEMENT; AUTOMATION; MACROMOLECULAR CRYSTALLOGRAPHY; STRUCTURE SIMILARITY; MODELING; PHENIX; MORPHING An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Å radius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Å r.m.s.d. representing 65–100% of the structure. text/html Improved crystallographic models through iterated local density-guided model deformation and reciprocal-space refinement text 7 68 2012-07-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 861 med@iucr.org 870 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5200 Small-angle scattering is becoming a mainstream technique for structural molecular biology. As such, it is important to establish guidelines for publication that will ensure that there is adequate reporting of the data and its treatment so that reviewers and readers can independently assess the quality of the data and the basis for any interpretations presented. This article presents a set of preliminary guidelines that emerged after consultation with the IUCr Commission on Small-Angle Scattering and other experts in the field and discusses the rationale for their application. At the 2011 Congress of the IUCr in Madrid, the Commission on Journals agreed to adopt these preliminary guidelines for the presentation of biomolecular structures from small-angle scattering data in IUCr publications. Here, these guidelines are outlined and the reasons for standardizing the way in which small-angle scattering data are presented. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Jacques, D.A. Guss, J.M. Svergun, D.I. Trewhella, J. 2012-06-01 doi:10.1107/S0907444912012073 International Union of Crystallography This article presents a set of preliminary publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution, and discusses the rationale for their application. en SMALL-ANGLE SCATTERING; PUBLICATION GUIDELINES Small-angle scattering is becoming a mainstream technique for structural molecular biology. As such, it is important to establish guidelines for publication that will ensure that there is adequate reporting of the data and its treatment so that reviewers and readers can independently assess the quality of the data and the basis for any interpretations presented. This article presents a set of preliminary guidelines that emerged after consultation with the IUCr Commission on Small-Angle Scattering and other experts in the field and discusses the rationale for their application. At the 2011 Congress of the IUCr in Madrid, the Commission on Journals agreed to adopt these preliminary guidelines for the presentation of biomolecular structures from small-angle scattering data in IUCr publications. Here, these guidelines are outlined and the reasons for standardizing the way in which small-angle scattering data are presented. text/html Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution text 6 68 2012-06-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 scientific comment 620 med@iucr.org 626 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5182 Tetartohedral crystal twinning is discussed as a particular case of (pseudo)merohedral twinning when the number of twinned domains is four. Tetartohedrally twinned crystals often possess pseudosymmetry, with the rotational part of the pseudo­symmetry operators coinciding with the twinning operators. Tetartohedrally twinned structures from the literature are reviewed and the recent structure determination of tetartohedrally twinned triclinic crystals of human complement factor I is discussed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Roversi, P. Blanc, E. Johnson, S. Lea, S.M. 2012-04-01 doi:10.1107/S0907444912006737 International Union of Crystallography A review of published tetartohedrally twinned macromolecular structures is presented, together with details of the recent structure determination of triclinic tetartohedrally twinned crystals of human complement factor I. en TETARTOHEDRAL TWINNING Tetartohedral crystal twinning is discussed as a particular case of (pseudo)merohedral twinning when the number of twinned domains is four. Tetartohedrally twinned crystals often possess pseudosymmetry, with the rotational part of the pseudo­symmetry operators coinciding with the twinning operators. Tetartohedrally twinned structures from the literature are reviewed and the recent structure determination of tetartohedrally twinned triclinic crystals of human complement factor I is discussed. text/html Tetartohedral twinning could happen to you too text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 418 med@iucr.org 424 http://scripts.iucr.org/cgi-bin/paper?xb5044 Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-­G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Selmer, M. Gao, Y.-G. Weixlbaumer, A. Ramakrishnan, V. 2012-05-01 doi:10.1107/S0907444912006348 International Union of Crystallography Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. en RIBOSOME; GTPASE; ENGINEERING Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-­G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors. text/html Ribosome engineering to promote new crystal forms text 5 68 2012-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 578 med@iucr.org 583 1399-0047 http://scripts.iucr.org/cgi-bin/paper?tz5004 During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-­rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Owen, R.L. Yorke, B.A. Pearson, A.R. 2012-05-01 doi:10.1107/S0907444912002946 International Union of Crystallography Macromolecular crystals luminesce during X-ray irradiation. This luminescence, and how it changes as a function of absorbed dose, has been characterized for several different proteins. en X-RAY-EXCITED OPTICAL LUMINESCENCE; RADIATION DAMAGE; MACROMOLECULAR CRYSTALLOGRAPHY During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-­rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals. text/html X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection text 5 68 2012-05-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 505 med@iucr.org 510 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5247 Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user's additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user's library and generate new monomer entries is described, as well as new entries for links and associated modifications. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Lebedev, A.A. Young, P. Isupov, M.N. Moroz, O.V. Vagin, A.A. Murshudov, G.N. 2012-04-01 doi:10.1107/S090744491200251X International Union of Crystallography The CCP4 template-restraint library defines restraints for biopolymers, their modifications and ligands that are used in macromolecular structure refinement. JLigand is a graphical editor for generating descriptions of new ligands and covalent linkages. en MACROMOLECULAR REFINEMENT; RESTRAINT LIBRARY; MOLECULAR GRAPHICS Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user's additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user's library and generate new monomer entries is described, as well as new entries for links and associated modifications. text/html JLigand: a graphical tool for the CCP4 template-restraint library text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 431 med@iucr.org 440 http://scripts.iucr.org/cgi-bin/paper?me0469 Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Steiner, R.A. Rupp, B. 2012-04-01 doi:10.1107/S0907444912002090 International Union of Crystallography An introduction to the proceedings of the CCP4 Study Weekend held at the University of Warwick on the 6–7 January 2011. en CCP4 STUDY WEEKEND text/html Model building, refinement and validation text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2012 International Union of Crystallography 0907-4449 issue preface 325 med@iucr.org 327 http://scripts.iucr.org/cgi-bin/paper?mh5057 Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Nagae, T. Kawamura, T. Chavas, L.M.G. Niwa, K. Hasegawa, M. Kato, C. Watanabe, N. 2012-03-01 doi:10.1107/S0907444912001862 International Union of Crystallography Structures of 3-­isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. en HIGH-PRESSURE PROTEIN CRYSTALLOGRAPHY; PRESSURE DENATURATION; WATER PENETRATION; 3-ISOPROPYLMALATE DEHYDROGENASE Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH. text/html High-pressure-induced water penetration into 3-­isopropylmalate dehydrogenase text 68 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-03-01 300 research papers 0907-4449 med@iucr.org 309 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5180 phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Afonine, P.V. Grosse-Kunstleve, R.W. Echols, N. Headd, J.J. Moriarty, N.W. Mustyakimov, M. Terwilliger, T.C. Urzhumtsev, A. Zwart, P.H. Adams, P.D. 2012-04-01 doi:10.1107/S0907444912001308 International Union of Crystallography phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods. en STRUCTURE REFINEMENT; PHENIX; JOINT X-RAY/NEUTRON REFINEMENT; MAXIMUM LIKELIHOOD; TLS; SIMULATED ANNEALING; SUBATOMIC RESOLUTION; REAL-SPACE REFINEMENT; TWINNING; NCS phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods. text/html Towards automated crystallographic structure refinement with phenix.refine text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 352 med@iucr.org 367 http://scripts.iucr.org/cgi-bin/paper?dz5246 Deformable elastic network (DEN) restraints have proved to be a powerful tool for refining structures from low-resolution X-ray crystallographic data sets. Unfortunately, optimal refinement using DEN restraints requires extensive calculations and is often hindered by a lack of access to sufficient computational resources. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements in parallel on the Open Science Grid, the US cyberinfrastructure. Access to the grid is provided through a simple and intuitive web interface integrated into the SBGrid Science Portal. Using this portal, refinements combined with full parameter optimization that would take many thousands of hours on standard computational resources can now be completed in several hours. An example of the successful application of DEN restraints to the human Notch1 transcriptional complex using the grid resource, and summaries of all submitted refinements, are presented as justification. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 O'Donovan, D.J. Stokes-Rees, I. Nam, Y. Blacklow, S.C. Schroder, G.F. Brunger, A.T. Sliz, P. 2012-03-01 doi:10.1107/S0907444912001163 International Union of Crystallography The deformable elastic network (DEN) method for reciprocal-space crystallographic refinement improves crystal structures, especially at resolutions lower than 3.5 Å. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements. en DEFORMABLE ELASTIC NETWORK RESTRAINTS; LOW-RESOLUTION REFINEMENT; DEN REFINEMENT Deformable elastic network (DEN) restraints have proved to be a powerful tool for refining structures from low-resolution X-ray crystallographic data sets. Unfortunately, optimal refinement using DEN restraints requires extensive calculations and is often hindered by a lack of access to sufficient computational resources. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements in parallel on the Open Science Grid, the US cyberinfrastructure. Access to the grid is provided through a simple and intuitive web interface integrated into the SBGrid Science Portal. Using this portal, refinements combined with full parameter optimization that would take many thousands of hours on standard computational resources can now be completed in several hours. An example of the successful application of DEN restraints to the human Notch1 transcriptional complex using the grid resource, and summaries of all submitted refinements, are presented as justification. text/html A grid-enabled web service for low-resolution crystal structure refinement text 68 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-03-01 261 research papers 0907-4449 med@iucr.org 267 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5173 The process of building and refining crystal structures of nucleic acids, although similar to that for proteins, has some peculiarities that give rise to both various complications and various benefits. Although conventional isomorphous replacement phasing techniques are typically used to generate an experimental electron-density map for the purposes of determining novel nucleic acid structures, it is also possible to couple the phasing and model-building steps to permit the solution of complex and novel RNA three-dimensional structures without the need for conventional heavy-atom phasing approaches. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Scott, W.G. 2012-04-01 doi:10.1107/S0907444912001084 International Union of Crystallography The challenges that arise in nucleic acid model building as a consequence of their simpler and more symmetric super-secondary structures are addressed. en NUCLEIC ACIDS; MODEL BUILDING; REFINEMENT The process of building and refining crystal structures of nucleic acids, although similar to that for proteins, has some peculiarities that give rise to both various complications and various benefits. Although conventional isomorphous replacement phasing techniques are typically used to generate an experimental electron-density map for the purposes of determining novel nucleic acid structures, it is also possible to couple the phasing and model-building steps to permit the solution of complex and novel RNA three-dimensional structures without the need for conventional heavy-atom phasing approaches. text/html Challenges and surprises that arise with nucleic acids during model building and refinement text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 441 med@iucr.org 445 http://scripts.iucr.org/cgi-bin/paper?ba5175 Coot is a molecular-graphics application primarily aimed to assist in model building and validation of biological macromolecules. Recently, tools have been added to work with small molecules. The newly incorporated tools for the manipulation and validation of ligands include interaction with PRODRG, subgraph isomorphism-based tools, representation of ligand chemistry, ligand fitting and analysis, and are described here. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Debreczeni, J.É. Emsley, P. 2012-04-01 doi:10.1107/S0907444912000200 International Union of Crystallography Coot is a molecular-graphics program designed to assist in the building of protein and other macromolecular models. The current state of ligand tools is presented. en COOT; LIGANDS Coot is a molecular-graphics application primarily aimed to assist in model building and validation of biological macromolecules. Recently, tools have been added to work with small molecules. The newly incorporated tools for the manipulation and validation of ligands include interaction with PRODRG, subgraph isomorphism-based tools, representation of ligand chemistry, ligand fitting and analysis, and are described here. text/html Handling ligands with Coot text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 425 med@iucr.org 430 http://scripts.iucr.org/cgi-bin/paper?ba5183 Two aspects of low-resolution macromolecular crystal structure analysis are considered: (i) the use of reference structures and structural units for provision of structural prior information and (ii) map sharpening in the presence of noise and the effects of Fourier series termination. The generation of interatomic distance restraints by ProSMART and their subsequent application in REFMAC5 is described. It is shown that the use of such external structural information can enhance the reliability of derived atomic models and stabilize refinement. The problem of map sharpening is considered as an inverse deblurring problem and is solved using Tikhonov regularizers. It is demonstrated that this type of map sharpening can automatically produce a map with more structural features whilst maintaining connectivity. Tests show that both of these directions are promising, although more work needs to be performed in order to further exploit structural information and to address the problem of reliable electron-density calculation. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Nicholls, R.A. Long, F. Murshudov, G.N. 2012-04-01 doi:10.1107/S090744491105606X International Union of Crystallography Low-resolution refinement tools implemented in REFMAC5 are described, including the use of external structural restraints, helical restraints and regularized anisotropic map sharpening. en LOW-RESOLUTION REFINEMENT; REFMAC5 Two aspects of low-resolution macromolecular crystal structure analysis are considered: (i) the use of reference structures and structural units for provision of structural prior information and (ii) map sharpening in the presence of noise and the effects of Fourier series termination. The generation of interatomic distance restraints by ProSMART and their subsequent application in REFMAC5 is described. It is shown that the use of such external structural information can enhance the reliability of derived atomic models and stabilize refinement. The problem of map sharpening is considered as an inverse deblurring problem and is solved using Tikhonov regularizers. It is demonstrated that this type of map sharpening can automatically produce a map with more structural features whilst maintaining connectivity. Tests show that both of these directions are promising, although more work needs to be performed in order to further exploit structural information and to address the problem of reliable electron-density calculation. text/html Low-resolution refinement tools in REFMAC5 text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 404 med@iucr.org 417 http://scripts.iucr.org/cgi-bin/paper?ba5178 Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct `target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -­target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Smart, O.S. Womack, T.O. Flensburg, C. Keller, P. Paciorek, W. Sharff, A. Vonrhein, C. Bricogne, G. 2012-04-01 doi:10.1107/S0907444911056058 International Union of Crystallography Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled. en BUSTER; NCS RESTRAINTS; TARGET-STRUCTURE RESTRAINTS; LOCAL STRUCTURAL SIMILARITY RESTRAINTS Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct `target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -­target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand. text/html Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 368 med@iucr.org 380 http://scripts.iucr.org/cgi-bin/paper?ba5181 Since its release in September 2009, the structure-solution program ARCIMBOLDO, based on the combination of locating small model fragments such as polyalanine α-helices with density modification with the program SHELXE in a multisolution frame, has evolved to incorporate other sources of stereochemical or experimental information. Fragments that are more sophisticated than the ubiquitous main-chain α-­helix can be proposed by modelling side chains onto the main chain or extracted from low-homology models, as locally their structure may be similar enough to the unknown one even if the conventional molecular-replacement approach has been unsuccessful. In such cases, the program may test a set of alternative models in parallel against a specified figure of merit and proceed with the selected one(s). Experimental information can be incorporated in three ways: searching within ARCIMBOLDO for an anomalous fragment against anomalous differences or MAD data or finding model fragments when an anomalous substructure has been determined with another program such as SHELXD or is subsequently located in the anomalous Fourier map calculated from the partial fragment phases. Both sources of information may be combined in the expansion process. In all these cases the key is to control the workflow to maximize the chances of success whilst avoiding the creation of an intractable number of parallel processes. A GUI has been implemented to aid the setup of suitable strategies within the various typical scenarios. In the present work, the practical application of ARCIMBOLDO within each of these scenarios is described through the distributed test cases. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Rodríguez, D. Sammito, M. Meindl, K. de Ilarduya, I.M. Potratz, M. Sheldrick, G.M. Usón, I. 2012-04-01 doi:10.1107/S0907444911056071 International Union of Crystallography ARCIMBOLDO combines the location of small fragments with Phaser and density modification with SHELXE of all possible Phaser solutions. Its uses are explained and illustrated through practical test cases. en ARCIMBOLDO; FRAGMENT SEARCH; PHASER; DENSITY MODIFICATION; MULTI-SOLUTION PHASING; SHELXE Since its release in September 2009, the structure-solution program ARCIMBOLDO, based on the combination of locating small model fragments such as polyalanine α-helices with density modification with the program SHELXE in a multisolution frame, has evolved to incorporate other sources of stereochemical or experimental information. Fragments that are more sophisticated than the ubiquitous main-chain α-­helix can be proposed by modelling side chains onto the main chain or extracted from low-homology models, as locally their structure may be similar enough to the unknown one even if the conventional molecular-replacement approach has been unsuccessful. In such cases, the program may test a set of alternative models in parallel against a specified figure of merit and proceed with the selected one(s). Experimental information can be incorporated in three ways: searching within ARCIMBOLDO for an anomalous fragment against anomalous differences or MAD data or finding model fragments when an anomalous substructure has been determined with another program such as SHELXD or is subsequently located in the anomalous Fourier map calculated from the partial fragment phases. Both sources of information may be combined in the expansion process. In all these cases the key is to control the workflow to maximize the chances of success whilst avoiding the creation of an intractable number of parallel processes. A GUI has been implemented to aid the setup of suitable strategies within the various typical scenarios. In the present work, the practical application of ARCIMBOLDO within each of these scenarios is described through the distributed test cases. text/html Practical structure solution with ARCIMBOLDO text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 336 med@iucr.org 343 http://scripts.iucr.org/cgi-bin/paper?ba5174 Developments of the PDB_REDO procedure that combine re-refinement and rebuilding within a unique decision-making framework to improve structures in the PDB are presented. PDB_REDO uses a variety of existing and custom-built software modules to choose an optimal refinement protocol (e.g. anisotropic, isotropic or overall B-factor refinement, TLS model) and to optimize the geometry versus data-refinement weights. Next, it proceeds to rebuild side chains and peptide planes before a final optimization round. PDB_REDO works fully automatically without the need for intervention by a crystallographic expert. The pipeline was tested on 12 000 PDB entries and the great majority of the test cases improved both in terms of crystallographic criteria such as Rfree and in terms of widely accepted geometric validation criteria. It is concluded that PDB_REDO is useful to update the otherwise `static' structures in the PDB to modern crystallographic standards. The publically available PDB_REDO database provides better model statistics and contributes to better refinement and validation targets. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Joosten, R.P. Joosten, K. Murshudov, G.N. Perrakis, A. 2012-04-01 doi:10.1107/S0907444911054515 International Union of Crystallography The decision-making algorithms and software used in PDB_REDO to re-refine and rebuild crystallographic protein structures in the PDB are presented and discussed. en VALIDATION; REFINEMENT; MODEL BUILDING; AUTOMATION; PDB Developments of the PDB_REDO procedure that combine re-refinement and rebuilding within a unique decision-making framework to improve structures in the PDB are presented. PDB_REDO uses a variety of existing and custom-built software modules to choose an optimal refinement protocol (e.g. anisotropic, isotropic or overall B-factor refinement, TLS model) and to optimize the geometry versus data-refinement weights. Next, it proceeds to rebuild side chains and peptide planes before a final optimization round. PDB_REDO works fully automatically without the need for intervention by a crystallographic expert. The pipeline was tested on 12 000 PDB entries and the great majority of the test cases improved both in terms of crystallographic criteria such as Rfree and in terms of widely accepted geometric validation criteria. It is concluded that PDB_REDO is useful to update the otherwise `static' structures in the PDB to modern crystallographic standards. The publically available PDB_REDO database provides better model statistics and contributes to better refinement and validation targets. text/html PDB_REDO: constructive validation, more than just looking for errors text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 484 med@iucr.org 496 http://scripts.iucr.org/cgi-bin/paper?cb5004 Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melano­gaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Klejnot, M. Kozielski, F. 2012-02-01 doi:10.1107/S0907444911053042 International Union of Crystallography The human Kif7 motor domain structure provides insights into a kinesin of medical significance. en KIF7; KIF27; HUMAN KINESINS; HEDGEHOG SIGNALLING Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melano­gaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance. text/html Structural insights into human Kif7, a kinesin involved in Hedgehog signalling text 2 68 2012-02-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 154 med@iucr.org 159 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5177 A novel method is presented for the automatic detection of noncrystallographic symmetry (NCS) in macromolecular crystal structure determination which does not require the derivation of molecular masks or the segmentation of density. It was found that throughout structure determination the NCS-related parts may be differently pronounced in the electron density. This often results in the modelling of molecular fragments of variable length and accuracy, especially during automated model-building procedures. These fragments were used to identify NCS relations in order to aid automated model building and refinement. In a number of test cases higher completeness and greater accuracy of the obtained structures were achieved, specifically at a crystallo­graphic resolution of 2.3 Å or poorer. In the best case, the method allowed the building of up to 15% more residues automatically and a tripling of the average length of the built fragments. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Wiegels, T. Lamzin, V.S. 2012-04-01 doi:10.1107/S0907444911050712 International Union of Crystallography Noncrystallographic symmetry is automatically detected and used to achieve higher completeness and greater accuracy of automatically built protein structures at resolutions of 2.3 Å or poorer. en NONCRYSTALLOGRAPHIC SYMMETRY; AUTOMATED MODEL BUILDING A novel method is presented for the automatic detection of noncrystallographic symmetry (NCS) in macromolecular crystal structure determination which does not require the derivation of molecular masks or the segmentation of density. It was found that throughout structure determination the NCS-related parts may be differently pronounced in the electron density. This often results in the modelling of molecular fragments of variable length and accuracy, especially during automated model-building procedures. These fragments were used to identify NCS relations in order to aid automated model building and refinement. In a number of test cases higher completeness and greater accuracy of the obtained structures were achieved, specifically at a crystallo­graphic resolution of 2.3 Å or poorer. In the best case, the method allowed the building of up to 15% more residues automatically and a tripling of the average length of the built fragments. text/html Use of noncrystallographic symmetry for automated model building at medium to low resolution text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 446 med@iucr.org 453 http://scripts.iucr.org/cgi-bin/paper?be5192 Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicaemia. The pathogen is a leading cause of nosocomial acquired infections, a problem that is exacerbated by the existence of methicillin- and glycopeptide antibiotic-resistant strains which can be challenging to treat. Alanine racemase (Alr) is a pyridoxal-5′-phosphate-dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As d-alanine is an essential component of the bacterial cell-wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. The crystal structure of S. aureus alanine racemase (AlrSas), the sequence of which corresponds to that from the highly antibiotic-resistant Mu50 strain, has been solved to 2.15 Å resolution. Comparison of the AlrSas structure with those of various alanine racemases demonstrates a conserved overall fold, with the enzyme sharing most similarity to those from other Gram-positive bacteria. Structural examination indicates that the active-site binding pocket, dimer interface and active-site entryway of the enzyme are potential targets for structure-aided inhibitor design. Kinetic constants were calculated in this study and are reported here. The potential for a disulfide bond in this structure is noted. This structural and biochemical information provides a template for future structure-based drug-development efforts targeting AlrSas. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Scaletti, E.R. Luckner, S.R. Krause, K.L. 2012-01-01 doi:10.1107/S0907444911050682 International Union of Crystallography The tertiary structure and kinetic properties of alanine racemase from Staphylococcus aureus are described and compared to other related alanine racemase structures. en ALANINE RACEMASE; STAPHYLOCOCCUS AUREUS Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicaemia. The pathogen is a leading cause of nosocomial acquired infections, a problem that is exacerbated by the existence of methicillin- and glycopeptide antibiotic-resistant strains which can be challenging to treat. Alanine racemase (Alr) is a pyridoxal-5′-phosphate-dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As d-alanine is an essential component of the bacterial cell-wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. The crystal structure of S. aureus alanine racemase (AlrSas), the sequence of which corresponds to that from the highly antibiotic-resistant Mu50 strain, has been solved to 2.15 Å resolution. Comparison of the AlrSas structure with those of various alanine racemases demonstrates a conserved overall fold, with the enzyme sharing most similarity to those from other Gram-positive bacteria. Structural examination indicates that the active-site binding pocket, dimer interface and active-site entryway of the enzyme are potential targets for structure-aided inhibitor design. Kinetic constants were calculated in this study and are reported here. The potential for a disulfide bond in this structure is noted. This structural and biochemical information provides a template for future structure-based drug-development efforts targeting AlrSas. text/html Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50) text 68 1 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2012-01-01 82 research papers 0907-4449 med@iucr.org 92 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5176 There is an increasing realisation that the quality of the biomacromolecular structures deposited in the Protein Data Bank (PDB) archive needs to be assessed critically using established and powerful validation methods. The Worldwide Protein Data Bank (wwPDB) organization has convened several Validation Task Forces (VTFs) to advise on the methods and standards that should be used to validate all of the entries already in the PDB as well as all structures that will be deposited in the future. The recommendations of the X-ray VTF are currently being implemented in a software pipeline. Here, ongoing work on this pipeline is briefly described as well as ways in which validation-related information could be presented to users of structural data. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gore, S. Velankar, S. Kleywegt, G.J. 2012-04-01 doi:10.1107/S0907444911050359 International Union of Crystallography The implementation of a validation pipeline, based on community recommendations, for future depositions of X-ray crystal structures in the Protein Data Bank is described. en VALIDATION; DATA ARCHIVES; BIOMACROMOLECULAR STRUCTURE; PDB There is an increasing realisation that the quality of the biomacromolecular structures deposited in the Protein Data Bank (PDB) archive needs to be assessed critically using established and powerful validation methods. The Worldwide Protein Data Bank (wwPDB) organization has convened several Validation Task Forces (VTFs) to advise on the methods and standards that should be used to validate all of the entries already in the PDB as well as all structures that will be deposited in the future. The recommendations of the X-ray VTF are currently being implemented in a software pipeline. Here, ongoing work on this pipeline is briefly described as well as ways in which validation-related information could be presented to users of structural data. text/html Implementing an X-ray validation pipeline for the Protein Data Bank text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 478 med@iucr.org 483 http://scripts.iucr.org/cgi-bin/paper?wd5161 The data-collection parameters used in a macromolecular diffraction experiment have a strong impact on data quality. A careful choice of parameters leads to better data and can make the difference between success and failure in phasing attempts, and will also result in a more accurate atomic model. The selection of parameters has to account for the application of the data in various phasing methods or high-resolution refinement. Furthermore, experimental factors such as crystal characteristics, available experiment time and the properties of the X-ray source and detector have to be considered. For many years, CCD detectors have been the prevalent type of detectors used in macromolecular crystallography. Recently, hybrid pixel X-ray detectors that operate in single-photon-counting mode have become available. These detectors have fundamentally different characteristics compared with CCD detectors and different data-collection strategies should be applied. Fine ϕ-slicing is a strategy that is particularly well suited to hybrid pixel detectors because of the fast readout time and the absence of readout noise. A large number of data sets were systematically collected from crystals of four different proteins in order to investigate the benefit of fine ϕ-­slicing on data quality with a noise-free detector. The results show that fine ϕ-slicing can substantially improve scaling statistics and anomalous signal provided that the rotation angle is comparable to half the crystal mosaicity. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Mueller, M. Wang, M. Schulze-Briese, C. 2012-01-01 doi:10.1107/S0907444911049833 International Union of Crystallography Fine ϕ-slicing substantially improves scaling statistics and anomalous signal for diffraction data collection with hybrid pixel detectors. en DIFFRACTION DATA COLLECTION; DATA-COLLECTION STRATEGIES; DETECTORS; HYBRID PIXEL DETECTOR; SINGLE-PHOTON COUNTING The data-collection parameters used in a macromolecular diffraction experiment have a strong impact on data quality. A careful choice of parameters leads to better data and can make the difference between success and failure in phasing attempts, and will also result in a more accurate atomic model. The selection of parameters has to account for the application of the data in various phasing methods or high-resolution refinement. Furthermore, experimental factors such as crystal characteristics, available experiment time and the properties of the X-ray source and detector have to be considered. For many years, CCD detectors have been the prevalent type of detectors used in macromolecular crystallography. Recently, hybrid pixel X-ray detectors that operate in single-photon-counting mode have become available. These detectors have fundamentally different characteristics compared with CCD detectors and different data-collection strategies should be applied. Fine ϕ-slicing is a strategy that is particularly well suited to hybrid pixel detectors because of the fast readout time and the absence of readout noise. A large number of data sets were systematically collected from crystals of four different proteins in order to investigate the benefit of fine ϕ-­slicing on data quality with a noise-free detector. The results show that fine ϕ-slicing can substantially improve scaling statistics and anomalous signal provided that the rotation angle is comparable to half the crystal mosaicity. text/html Optimal fine ϕ-slicing for single-photon-counting pixel detectors text 68 1 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2012-01-01 42 research papers 0907-4449 med@iucr.org 56 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5170 Situs is a modular program package for the multi-scale modeling of atomic resolution structures and low-resolution biophysical data from electron microscopy, tomography or small-angle X-ray scattering. This article provides an overview of recent developments in the Situs package, with an emphasis on workflows and conventions that are important for practical applications. The modular design of the programs facilitates scripting in the bash shell that allows specific programs to be combined in creative ways that go beyond the original intent of the developers. Several scripting-enabled functionalities, such as flexible transformations of data type, the use of symmetry constraints or the creation of two-dimensional projection images, are described. The processing of low-resolution biophysical maps in such workflows follows not only first principles but often relies on implicit conventions. Situs conventions related to map formats, resolution, correlation functions and feature detection are reviewed and summarized. The compatibility of the Situs workflow with CCP4 conventions and programs is discussed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Wriggers, W. 2012-04-01 doi:10.1107/S0907444911049791 International Union of Crystallography Recent developments of the Situs software suite for multi-scale modeling are reviewed. Typical workflows and conventions encountered during processing of biophysical data from electron microscopy, tomography or small-angle X-ray scattering are described. en SITUS; MULTI-RESOLUTION DATA Situs is a modular program package for the multi-scale modeling of atomic resolution structures and low-resolution biophysical data from electron microscopy, tomography or small-angle X-ray scattering. This article provides an overview of recent developments in the Situs package, with an emphasis on workflows and conventions that are important for practical applications. The modular design of the programs facilitates scripting in the bash shell that allows specific programs to be combined in creative ways that go beyond the original intent of the developers. Several scripting-enabled functionalities, such as flexible transformations of data type, the use of symmetry constraints or the creation of two-dimensional projection images, are described. The processing of low-resolution biophysical maps in such workflows follows not only first principles but often relies on implicit conventions. Situs conventions related to map formats, resolution, correlation functions and feature detection are reviewed and summarized. The compatibility of the Situs workflow with CCP4 conventions and programs is discussed. text/html Conventions and workflows for using Situs text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 344 med@iucr.org 351 http://scripts.iucr.org/cgi-bin/paper?ba5179 Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Brunger, A.T. Das, D. Deacon, A.M. Grant, J. Terwilliger, T.C. Read, R.J. Adams, P.D. Levitt, M. Schröder, G.F. 2012-04-01 doi:10.1107/S090744491104978X International Union of Crystallography DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols. en RECIPROCAL-SPACE REFINEMENT; DEN REFINEMENT; REAL-SPACE REFINEMENT; AUTOMATED MODEL BUILDING; SUCCINYL-DIAMINOPIMELATE DESUCCINYLASE Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence. text/html Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 391 med@iucr.org 403 http://scripts.iucr.org/cgi-bin/paper?gx9191 A correction is made to a citation in the article by Suzuki et al. (2011) [Acta Cryst. D67, 894–901]. Copyright (c) 2011 International Union of Crystallography urn:issn:0907-4449 Suzuki, K. Yang, S.-Y. Shimizu, S. Morishita, E.C. Jiang, J. Zhang, F. Hoque, M.M. Sato, Y. Tsunoda, M. Sekiguchi, T. Takénaka, A. 2011-12-01 doi:10.1107/S0907444911048633 International Union of Crystallography A correction to the paper by Suzuki et al. [(2011). Acta Cryst. D67, 894–901]. en CARBONIC ANHYDRASE; CHLAMYDOMONAS REINHARDTII; CORRIGENDUM A correction is made to a citation in the article by Suzuki et al. (2011) [Acta Cryst. D67, 894–901]. text/html The unique structure of carbonic anhydrase αCA1 from Chlamydomonas reinhardtii. Corrigendum text 67 12 Copyright (c) 2011 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2011-12-01 1077 addenda and errata 0907-4449 med@iucr.org 1077 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5171 Traditional methods for macromolecular refinement often have limited success at low resolution (3.0–3.5 Å or worse), producing models that score poorly on crystallographic and geometric validation criteria. To improve low-resolution refinement, knowledge from macromolecular chemistry and homology was used to add three new coordinate-restraint functions to the refinement program phenix.refine. Firstly, a `reference-model' method uses an identical or homologous higher resolution model to add restraints on torsion angles to the geometric target function. Secondly, automatic restraints for common secondary-structure elements in proteins and nucleic acids were implemented that can help to preserve the secondary-structure geometry, which is often distorted at low resolution. Lastly, we have implemented Ramachandran-based restraints on the backbone torsion angles. In this method, a ϕ,ψ term is added to the geometric target function to minimize a modified Ramachandran landscape that smoothly combines favorable peaks identified from non­redundant high-quality data with unfavorable peaks calculated using a clash-based pseudo-energy function. All three methods show improved MolProbity validation statistics, typically complemented by a lowered Rfree and a decreased gap between Rwork and Rfree. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Headd, J.J. Echols, N. Afonine, P.V. Grosse-Kunstleve, R.W. Chen, V.B. Moriarty, N.W. Richardson, D.C. Richardson, J.S. Adams, P.D. 2012-04-01 doi:10.1107/S0907444911047834 International Union of Crystallography Recent developments in PHENIX are reported that allow the use of reference-model torsion restraints, secondary-structure hydrogen-bond restraints and Ramachandran restraints for improved macromolecular refinement in phenix.refine at low resolution. en MACROMOLECULAR CRYSTALLOGRAPHY; LOW RESOLUTION; REFINEMENT; AUTOMATION Traditional methods for macromolecular refinement often have limited success at low resolution (3.0–3.5 Å or worse), producing models that score poorly on crystallographic and geometric validation criteria. To improve low-resolution refinement, knowledge from macromolecular chemistry and homology was used to add three new coordinate-restraint functions to the refinement program phenix.refine. Firstly, a `reference-model' method uses an identical or homologous higher resolution model to add restraints on torsion angles to the geometric target function. Secondly, automatic restraints for common secondary-structure elements in proteins and nucleic acids were implemented that can help to preserve the secondary-structure geometry, which is often distorted at low resolution. Lastly, we have implemented Ramachandran-based restraints on the backbone torsion angles. In this method, a ϕ,ψ term is added to the geometric target function to minimize a modified Ramachandran landscape that smoothly combines favorable peaks identified from non­redundant high-quality data with unfavorable peaks calculated using a clash-based pseudo-energy function. All three methods show improved MolProbity validation statistics, typically complemented by a lowered Rfree and a decreased gap between Rwork and Rfree. text/html Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 381 med@iucr.org 390 http://scripts.iucr.org/cgi-bin/paper?mn5005 Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homo­tetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Rao, V.A. Shepherd, S.M. English, G. Coulthurst, S.J. Hunter, W.N. 2011-12-01 doi:10.1107/S0907444911046300 International Union of Crystallography The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. en [BETA]-SANDWICH; GRAM-NEGATIVE PATHOGENS; LIPOPROTEINS; PROTEIN SECRETION; TRANSTHYRETIN; VIRULENCE Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homo­tetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners. text/html The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system text 67 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-12-01 1065 research papers 0907-4449 med@iucr.org 1072 1399-0047 http://scripts.iucr.org/cgi-bin/paper?me0464 The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected. Copyright (c) 2011 International Union of Crystallography urn:issn:0907-4449 Abdulmalik, O. Ghatge, M.S. Musayev, F.N. Parikh, A. Chen, Q. Yang, J. Nnamani, I. Danso-Danquah, R. Eseonu, D.N. Asakura, T. Abraham, D.J. Venitz, J. Safo, M.K. 2011-12-01 doi:10.1107/S0907444911045860 International Union of Crystallography A correction to the paper by Abdulmalik et al. [(2011), Acta Cryst. D67, 920–928]. en HEMOGLOBIN; OXYGEN AFFINITY; SICKLE-CELL DISEASE; POLYMERIZATION; T STATE; R2 STATE; CORRIGENDUM The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected. text/html Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin. Corrigendum text 67 12 Copyright (c) 2011 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2011-12-01 1076 addenda and errata 0907-4449 med@iucr.org 1076 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5169 Two developments in the process of automated protein model building in the Buccaneer software are presented. A general-purpose library for protein fragments of arbitrary size is described, with a highly optimized search method allowing the use of a larger database than in previous work. The problem of assembling an autobuilt model into complete chains is discussed. This involves the assembly of disconnected chain fragments into complete molecules and the use of the database of protein fragments in improving the model completeness. Assembly of fragments into molecules is a standard step in existing model-building software, but the methods have not received detailed discussion in the literature. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Cowtan, K. 2012-04-01 doi:10.1107/S0907444911039655 International Union of Crystallography Two developments in the process of automated protein model building in the Buccaneer software are described: the use of a database of protein fragments in improving the model completeness and the assembly of disconnected chain fragments into complete molecules. en MODEL BUILDING; DATABASES; BUCCANEER Two developments in the process of automated protein model building in the Buccaneer software are presented. A general-purpose library for protein fragments of arbitrary size is described, with a highly optimized search method allowing the use of a larger database than in previous work. The problem of assembling an autobuilt model into complete chains is discussed. This involves the assembly of disconnected chain fragments into complete molecules and the use of the database of protein fragments in improving the model completeness. Assembly of fragments into molecules is a standard step in existing model-building software, but the methods have not received detailed discussion in the literature. text/html Completion of autobuilt protein models using a database of protein fragments text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 328 med@iucr.org 335 http://scripts.iucr.org/cgi-bin/paper?lv5014 Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immuno­deficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the Cα deviations are large and the active-site `DTG' loop (here NTG) deviates up to 2.7 Å from the standard con­formation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gilski, M. Kazmierczyk, M. Krzywda, S. Zábranská, H. Cooper, S. Popović, Z. Khatib, F. DiMaio, F. Thompson, J. Baker, D. Pichová, I. Jaskolski, M. 2011-11-01 doi:10.1107/S0907444911035943 International Union of Crystallography The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. en MASON-PFIZER MONKEY VIRUS; HIV-1; RETROVIRAL PROTEASES; RETROPEPSINS; PROTEIN FOLDING; DIMERIZATION INHIBITION; DRUG-DESIGN TARGETS; AIDS Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immuno­deficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the Cα deviations are large and the active-site `DTG' loop (here NTG) deviates up to 2.7 Å from the standard con­formation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS. text/html High-resolution structure of a retroviral protease folded as a monomer text 67 11 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-11-01 907 research papers 0907-4449 med@iucr.org 914 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5235 The commonly used validation metrics for the local agreement of a structure model with the observed electron density, namely the real-space R (RSR) and the real-space correlation coefficient (RSCC), are reviewed. It is argued that the primary goal of all validation techniques is to verify the accuracy of the model, since precision is an inherent property of the crystal and the data. It is demonstrated that the principal weakness of both of the above metrics is their inability to distinguish the accuracy of the model from its precision. Furthermore, neither of these metrics in their usual implementation indicate the statistical significance of the result. The statistical properties of electron-density maps are reviewed and an improved alternative likelihood-based metric is suggested. This leads naturally to a χ2 significance test of the difference density using the real-space difference density Z score (RSZD). This is a metric purely of the local model accuracy, as required for effective model validation and structure optimization by practising crystallographers prior to submission of a structure model to the PDB. A new real-space observed density Z score (RSZO) is also proposed; this is a metric purely of the model precision, as a substitute for other precision metrics such as the B factor. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Tickle, I.J. 2012-04-01 doi:10.1107/S0907444911035918 International Union of Crystallography A likelihood-based metric for scoring the local agreement of a structure model with the observed electron density is described. en DIFFERENCE DENSITY; ELECTRON DENSITY; MODEL ACCURACY; MODEL PRECISION; REAL-SPACE R; REAL-SPACE CORRELATION COEFFICIENT; REAL-SPACE DIFFERENCE DENSITY Z SCORE; REAL-SPACE OBSERVED DENSITY Z SCORE; STRUCTURE VALIDATION The commonly used validation metrics for the local agreement of a structure model with the observed electron density, namely the real-space R (RSR) and the real-space correlation coefficient (RSCC), are reviewed. It is argued that the primary goal of all validation techniques is to verify the accuracy of the model, since precision is an inherent property of the crystal and the data. It is demonstrated that the principal weakness of both of the above metrics is their inability to distinguish the accuracy of the model from its precision. Furthermore, neither of these metrics in their usual implementation indicate the statistical significance of the result. The statistical properties of electron-density maps are reviewed and an improved alternative likelihood-based metric is suggested. This leads naturally to a χ2 significance test of the difference density using the real-space difference density Z score (RSZD). This is a metric purely of the local model accuracy, as required for effective model validation and structure optimization by practising crystallographers prior to submission of a structure model to the PDB. A new real-space observed density Z score (RSZO) is also proposed; this is a metric purely of the model precision, as a substitute for other precision metrics such as the B factor. text/html Statistical quality indicators for electron-density maps text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 454 med@iucr.org 467 http://scripts.iucr.org/cgi-bin/paper?dz5234 In choosing and refining any crystallographic structural model, there is tension between the desire to extract the most detailed information possible and the necessity to describe no more than what is justified by the observed data. A more complex model is not necessarily a better model. Thus, it is important to validate the choice of parameters as well as validating their refined values. One recurring task is to choose the best model for describing the displacement of each atom about its mean position. At atomic resolution one has the option of devoting six model parameters (a `thermal ellipsoid') to describe the displacement of each atom. At medium resolution one typically devotes at most one model parameter per atom to describe the same thing (a `B factor'). At very low resolution one cannot justify the use of even one parameter per atom. Furthermore, this aspect of the structure may be described better by an explicit model of bulk displacements, the most common of which is the translation/libration/screw (TLS) formalism, rather than by assigning some number of para­meters to each atom individually. One can sidestep this choice between atomic displacement parameters and TLS descriptions by including both treatments in the same model, but this is not always statistically justifiable. The choice of which treatment is best for a particular structure refinement at a particular resolution can be guided by general considerations of the ratio of model parameters to the number of observations and by specific statistics such as the Hamilton R-­factor ratio test. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Merritt, E.A. 2012-04-01 doi:10.1107/S0907444911028320 International Union of Crystallography A simple rule of thumb based on resolution is not adequate to identify the best treatment of atomic displacements in macromolecular structural models. The choice to use isotropic B factors, anisotropic B factors, TLS models or some combination of the three should be validated through statistical analysis of the model refinement. en ATOMIC DISPLACEMENTS; B FACTORS; TLS MODELS; MODEL PARAMETERS In choosing and refining any crystallographic structural model, there is tension between the desire to extract the most detailed information possible and the necessity to describe no more than what is justified by the observed data. A more complex model is not necessarily a better model. Thus, it is important to validate the choice of parameters as well as validating their refined values. One recurring task is to choose the best model for describing the displacement of each atom about its mean position. At atomic resolution one has the option of devoting six model parameters (a `thermal ellipsoid') to describe the displacement of each atom. At medium resolution one typically devotes at most one model parameter per atom to describe the same thing (a `B factor'). At very low resolution one cannot justify the use of even one parameter per atom. Furthermore, this aspect of the structure may be described better by an explicit model of bulk displacements, the most common of which is the translation/libration/screw (TLS) formalism, rather than by assigning some number of para­meters to each atom individually. One can sidestep this choice between atomic displacement parameters and TLS descriptions by including both treatments in the same model, but this is not always statistically justifiable. The choice of which treatment is best for a particular structure refinement at a particular resolution can be guided by general considerations of the ratio of model parameters to the number of observations and by specific statistics such as the Hamilton R-­factor ratio test. text/html To B or not to B: a question of resolution? text 4 68 2012-04-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 468 med@iucr.org 477 http://scripts.iucr.org/cgi-bin/paper?gx5187 RiVax is a recombinant protein that is currently under clinical development as part of a human vaccine to protect against ricin poisoning. RiVax includes ricin A-chain (RTA) residues 1–267 with two intentional amino-acid substitutions, V76M and Y80A, aimed at reducing toxicity. Here, the crystal structure of RiVax was solved to 2.1 Å resolution and it was shown that it is superposable with that of the ricin toxin A-chain from Ricinus communis with a root-mean-square deviation of 0.6 Å over 258 Cα atoms. The RiVax structure is also compared with the recently determined structure of another potential ricin-vaccine immunogen, RTA 1–33/44–198 R48C/T77C. Finally, the locations and solvent-exposure of two toxin-neutralizing B-cell epitopes were examined and it was found that these epitopes are within or near regions predicted to be involved in catalysis. The results demonstrate the composition of the RiVax clinical material and will guide ongoing protein-engineering strategies to develop improved immunogens. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Legler, P.M. Brey, R.N. Smallshaw, J.E. Vitetta, E.S. Millard, C.B. 2011-09-01 doi:10.1107/S0907444911026771 International Union of Crystallography The X-ray crystal structure (at 2.1 Å resolution) of an immunogen under development as part of a ricin vaccine for humans is presented and structure-based analysis of the results was conducted with respect to related proteins and the known determinants for inducing or suppressing the protective immune response. en RICIN; RIBOSOME-INACTIVATING PROTEINS; PROTEIN ENGINEERING; IMMUNOGENS; RIVAX; B-CELL EPITOPES RiVax is a recombinant protein that is currently under clinical development as part of a human vaccine to protect against ricin poisoning. RiVax includes ricin A-chain (RTA) residues 1–267 with two intentional amino-acid substitutions, V76M and Y80A, aimed at reducing toxicity. Here, the crystal structure of RiVax was solved to 2.1 Å resolution and it was shown that it is superposable with that of the ricin toxin A-chain from Ricinus communis with a root-mean-square deviation of 0.6 Å over 258 Cα atoms. The RiVax structure is also compared with the recently determined structure of another potential ricin-vaccine immunogen, RTA 1–33/44–198 R48C/T77C. Finally, the locations and solvent-exposure of two toxin-neutralizing B-cell epitopes were examined and it was found that these epitopes are within or near regions predicted to be involved in catalysis. The results demonstrate the composition of the RiVax clinical material and will guide ongoing protein-engineering strategies to develop improved immunogens. text/html Structure of RiVax: a recombinant ricin vaccine text 67 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-09-01 826 research papers 0907-4449 med@iucr.org 830 1399-0047 http://scripts.iucr.org/cgi-bin/paper?yt9032 A correction is made to a figure in the article by Porta et al. [(2011). Acta Cryst. D67, 628–638]. Copyright (c) 2011 International Union of Crystallography urn:issn:0907-4449 Porta, J. Lovelace, J.J. Schreurs, A.M.M. Kroon-Batenburg, L.M.J. Borgstahl, G.E.O. 2011-08-01 doi:10.1107/S0907444911026631 International Union of Crystallography A correction to the article by Porta et al. [(2011). Acta Cryst. D67, 628–638]. en MODULATION; INCOMMENSURATE; EVAL15; PROFILIN-ACTIN; CORRIGENDUM A correction is made to a figure in the article by Porta et al. [(2011). Acta Cryst. D67, 628–638]. text/html Processing incommensurately modulated protein diffraction data with Eval15. Corrigendum text 8 67 2011-08-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2011 International Union of Crystallography 0907-4449 addenda and errata 745 med@iucr.org 745 1399-0047 http://scripts.iucr.org/cgi-bin/paper?yt5034 Assemblies with helical symmetry can be conveniently formulated in many distinct ways. Here, a new convention is presented which unifies the two most commonly used helical systems for generating helical assemblies from asymmetric units determined by X-ray fibre diffraction and EM imaging. A helical assembly is viewed as being composed of identical repetitive units in a one- or two-dimensional lattice, named 1-­D and 2-D helical systems, respectively. The unification suggests that a new helical description with only four parameters [n1, n2, twist, rise], which is called the augmented 1-D helical system, can generate the complete set of helical arrangements, including coverage of helical discontinuities (seams). A unified four-parameter characterization implies similar parameters for similar assemblies, can eliminate errors in reproducing structures of helical assemblies and facilitates the generation of polymorphic ensembles from helical atomic models or EM density maps. Further, guidelines are provided for such a unique description that reflects the structural signature of an assembly, as well as rules for manipulating the helical symmetry presentation. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Tsai, C.-J. Nussinov, R. 2011-08-01 doi:10.1107/S0907444911024024 International Union of Crystallography A new representation of helical structure by four parameters, [n1, n2, twist, rise], is able to generate an entire helical construct from asymmetric units, including cases of helical assembly with a seam. en SYMMETRY; X-RAY FIBRE DIFFRACTION; EM DENSITY MAPS; HELICAL ASSEMBLIES Assemblies with helical symmetry can be conveniently formulated in many distinct ways. Here, a new convention is presented which unifies the two most commonly used helical systems for generating helical assemblies from asymmetric units determined by X-ray fibre diffraction and EM imaging. A helical assembly is viewed as being composed of identical repetitive units in a one- or two-dimensional lattice, named 1-­D and 2-D helical systems, respectively. The unification suggests that a new helical description with only four parameters [n1, n2, twist, rise], which is called the augmented 1-D helical system, can generate the complete set of helical arrangements, including coverage of helical discontinuities (seams). A unified four-parameter characterization implies similar parameters for similar assemblies, can eliminate errors in reproducing structures of helical assemblies and facilitates the generation of polymorphic ensembles from helical atomic models or EM density maps. Further, guidelines are provided for such a unique description that reflects the structural signature of an assembly, as well as rules for manipulating the helical symmetry presentation. text/html A unified convention for biological assemblies with helical symmetry text 8 67 2011-08-01 Acta Crystallographica Section D: Biological Crystallography http://creativecommons.org/licenses/by/2.0/uk 0907-4449 research papers 716 med@iucr.org 728 1399-0047 http://scripts.iucr.org/cgi-bin/paper?yt5033 The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Meinke, G. Phelan, P. Fradet-Turcotte, A. Archambault, J. Bullock, P.A. 2011-06-01 doi:10.1107/S0907444911014302 International Union of Crystallography With the aim of forming the `lock-washer' conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. en SV40 LARGE T ANTIGEN; ORIGIN-BINDING DOMAIN; DISULFIDES; OLIGOMERIZATION; DNA REPLICATION The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner. text/html Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain text 67 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-06-01 560 research papers 0907-4449 med@iucr.org 567 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5225 The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kisselman, G. Qiu, W. Romanov, V. Thompson, C.M. Lam, R. Battaile, K.P. Pai, E.F. Chirgadze, N.Y. 2011-06-01 doi:10.1107/S0907444911011589 International Union of Crystallography The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. en PROTEIN CRYSTALLIZATION DEVICES; IN SITU X-RAY ANALYSIS; CRYSTALLIZATION; CRYSTAL VISUAL INSPECTION; DIFFRACTION DATA COLLECTION The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process. text/html X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection text 67 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-06-01 533 research papers 0907-4449 med@iucr.org 539 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5391 The Pi sampling method is derived from the incomplete factorial approach to macromolecular crystallization screen design. The resulting `Pi screens' have a modular distribution of a given set of up to 36 stock solutions. Maximally diverse conditions can be produced by taking into account the properties of the chemicals used in the formulation and the concentrations of the corresponding solutions. The Pi sampling method has been implemented in a web-based application that generates screen formulations and recipes. It is particularly adapted to screens consisting of 96 different conditions. The flexibility and efficiency of Pi sampling is demonstrated by the crystallization of soluble proteins and of an integral membrane-protein sample. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gorrec, F. Palmer, C.M. Lebon, G. Warne, T. 2011-05-01 doi:10.1107/S0907444911008754 International Union of Crystallography Pi sampling, derived from the incomplete factorial approach, is an effort to maximize the diversity of macromolecular crystallization conditions and to facilitate the preparation of 96-condition initial screens. en MACROMOLECULAR CRYSTALLIZATION; INITIAL SCREEN FORMULATION; INCOMPLETE FACTORIAL APPROACH; MODULAR DISTRIBUTION; MEMBRANE-PROTEIN CRYSTALLIZATION; GPCR The Pi sampling method is derived from the incomplete factorial approach to macromolecular crystallization screen design. The resulting `Pi screens' have a modular distribution of a given set of up to 36 stock solutions. Maximally diverse conditions can be produced by taking into account the properties of the chemicals used in the formulation and the concentrations of the corresponding solutions. The Pi sampling method has been implemented in a web-based application that generates screen formulations and recipes. It is particularly adapted to screens consisting of 96 different conditions. The flexibility and efficiency of Pi sampling is demonstrated by the crystallization of soluble proteins and of an integral membrane-protein sample. text/html Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening text 67 5 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-05-01 463 research papers 0907-4449 med@iucr.org 470 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5171 Two errors in sugar stereochemistry in the structure of ristocetin A have been corrected and the structure has been re-refined. Copyright (c) 2011 International Union of Crystallography urn:issn:0907-4449 Nahoum, V. Spector, S. Loll, P. 2011-06-01 doi:10.1107/S0907444911008262 International Union of Crystallography A correction to the article by Nahoum et al. [(2009). Acta Cryst. D65, 832–838]. en RISTOCETIN A; BACTERIAL CELL-WALL MIMETIC; CORRIGENDUM Two errors in sugar stereochemistry in the structure of ristocetin A have been corrected and the structure has been re-refined. text/html Structure of ristocetin A in complex with a bacterial cell-wall mimetic. Corrigendum. text 67 6 Copyright (c) 2011 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2011-06-01 592 addenda and errata 0907-4449 med@iucr.org 592 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5164 The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Morris, C. Pajon, A. Griffiths, S.L. Daniel, E. Savitsky, M. Lin, B. Diprose, J.M. Wilter da Silva, A. Pilicheva, K. Troshin, P. van Niekerk, J. Isaacs, N. Naismith, J. Nave, C. Blake, R. Wilson, K.S. Stuart, D.I. Henrick, K. Esnouf, R.M. 2011-04-01 doi:10.1107/S0907444911007943 International Union of Crystallography The Protein Information Management System (PiMS) is described together with a discussion of how its features make it well suited to laboratories of all sizes. en LABORATORY INFORMATION-MANAGEMENT SYSTEMS; PROTEIN PRODUCTION; JAVA WEB APPLICATIONS; DATA MANAGEMENT AND DATABASES The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service. text/html The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 249 research papers 0907-4449 med@iucr.org 260 http://scripts.iucr.org/cgi-bin/paper?xb5027 The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-­ray crystallography to 2.7 Å resolution. HLA-A3 is a predisposing allele for multiple sclerosis (MS), an auto­immune disease of the central nervous system. The KLIETYFSK peptide is a naturally processed epitope of proteolipid protein, a myelin protein and candidate target for immune-mediated myelin destruction in MS. Comparison of the structure of HLA-A3 with that of HLA-A2, an MHC class I molecule which is protective against MS, indicates that both MHC class I molecules present very similar faces for T-cell receptor recognition whilst differing in the specificity of their peptide-binding grooves. These characteristics may underlie the opposing (predisposing versus protective) associations that they exhibit both in humans and in mouse models of MS-­like disease. Furthermore, subtle alterations within the peptide-binding groove of HLA-A3 and other A3-like MHC class I molecules, members of the so-called A3 superfamily, may be sufficient to alter their presentation of autoantigen peptides such as KLIETYFSK. This in turn may modulate their contribution to the associated risk of autoimmune disease. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 McMahon, R.M. Friis, L. Siebold, C. Friese, M.A. Fugger, L. Jones, E.Y. 2011-05-01 doi:10.1107/S0907444911007888 International Union of Crystallography The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-­ray crystallography to 2.7 Å resolution. en HLA-A*0301; MHC; MULTIPLE SCLEROSIS; AUTOIMMUNE DISEASE The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-­ray crystallography to 2.7 Å resolution. HLA-A3 is a predisposing allele for multiple sclerosis (MS), an auto­immune disease of the central nervous system. The KLIETYFSK peptide is a naturally processed epitope of proteolipid protein, a myelin protein and candidate target for immune-mediated myelin destruction in MS. Comparison of the structure of HLA-A3 with that of HLA-A2, an MHC class I molecule which is protective against MS, indicates that both MHC class I molecules present very similar faces for T-cell receptor recognition whilst differing in the specificity of their peptide-binding grooves. These characteristics may underlie the opposing (predisposing versus protective) associations that they exhibit both in humans and in mouse models of MS-­like disease. Furthermore, subtle alterations within the peptide-binding groove of HLA-A3 and other A3-like MHC class I molecules, members of the so-called A3 superfamily, may be sufficient to alter their presentation of autoantigen peptides such as KLIETYFSK. This in turn may modulate their contribution to the associated risk of autoimmune disease. text/html Structure of HLA-A*0301 in complex with a peptide of proteolipid protein: insights into the role of HLA-­A alleles in susceptibility to multiple sclerosis text 67 5 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-05-01 447 research papers 0907-4449 med@iucr.org 454 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5162 Macromolecular crystallography relies on the availability and quality of single crystals; these are typically obtained through extensive screening, which has a very low intrinsic success rate. Crystallization is not a completely stochastic process and many proteins do not succumb to crystallization because of specific microscopic features of their molecular surfaces. It follows that rational surface engineering through site-directed mutagenesis should allow a systematic and significant improvement in crystallization success rates. Here, one such established strategy, surface-entropy reduction (SER), is discussed, including its successes, limitations and possible future developments. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Derewenda, Z.S. 2011-04-01 doi:10.1107/S0907444911007797 International Union of Crystallography Protein surface engineering is increasingly used as a routine tool to enhance the crystallization propensity of proteins. Future possibilities include the use of multi-site protein variants, rational modulation of solubility and the development of strategies to tackle membrane proteins. en MACROMOLECULAR CRYSTALLOGRAPHY; CRYSTALS; CRYSTALLIZATION; PROTEIN ENGINEERING; CRYSTAL CONTACTS Macromolecular crystallography relies on the availability and quality of single crystals; these are typically obtained through extensive screening, which has a very low intrinsic success rate. Crystallization is not a completely stochastic process and many proteins do not succumb to crystallization because of specific microscopic features of their molecular surfaces. It follows that rational surface engineering through site-directed mutagenesis should allow a systematic and significant improvement in crystallization success rates. Here, one such established strategy, surface-entropy reduction (SER), is discussed, including its successes, limitations and possible future developments. text/html It's all in the crystals… text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 243 research papers 0907-4449 med@iucr.org 248 http://scripts.iucr.org/cgi-bin/paper?ba5166 A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Vonrhein, C. Flensburg, C. Keller, P. Sharff, A. Smart, O. Paciorek, W. Womack, T. Bricogne, G. 2011-04-01 doi:10.1107/S0907444911007773 International Union of Crystallography Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described. en AUTOPROC; DATA PROCESSING A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats. text/html Data processing and analysis with the autoPROC toolbox text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 293 research papers 0907-4449 med@iucr.org 302 http://scripts.iucr.org/cgi-bin/paper?ba5168 The measurement of X-ray diffraction data from macro­molecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Evans, G. Axford, D. Owen, R.L. 2011-04-01 doi:10.1107/S0907444911007608 International Union of Crystallography Thoughts about the decisions made in designing macromolecular X-ray crystallography experiments at synchrotron beamlines are presented. en MACROMOLECULAR CRYSTALLOGRAPHY; MICROCRYSTALLOGRAPHY; X-RAY BEAMLINES; SYNCHROTRON RADIATION The measurement of X-ray diffraction data from macro­molecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals. text/html The design of macromolecular crystallography diffraction experiments text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 261 research papers 0907-4449 med@iucr.org 270 http://scripts.iucr.org/cgi-bin/paper?ba5167 Molecular replacement is one of the key methods used to solve the problem of determining the phases of structure factors in protein structure solution from X-ray image diffraction data. Its success rate has been steadily improving with the development of improved software methods and the increasing number of structures available in the PDB for use as search models. Despite this, in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues it can be a difficult and time-consuming chore to isolate and prepare the best search model for molecular replacement. MrBUMP and BALBES are two recent developments from CCP4 that have been designed to automate and speed up the process of determining and preparing the best search models and putting them through molecular replacement. Their intention is to provide the user with a broad set of results using many search models and to highlight the best of these for further processing. An overview of both programs is presented along with a description of how best to use them, citing case studies and the results of large-scale testing of the software. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Keegan, R.M. Long, F. Fazio, V.J. Winn, M.D. Murshudov, G.N. Vagin, A.A. 2011-04-01 doi:10.1107/S0907444911007530 International Union of Crystallography The automated pipelines for molecular replacement MrBUMP and BALBES are reviewed, with an emphasis on understanding their output. Conclusions are drawn from their performance in extensive trials. en MRBUMP; BALBES; MOLECULAR REPLACEMENT Molecular replacement is one of the key methods used to solve the problem of determining the phases of structure factors in protein structure solution from X-ray image diffraction data. Its success rate has been steadily improving with the development of improved software methods and the increasing number of structures available in the PDB for use as search models. Despite this, in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues it can be a difficult and time-consuming chore to isolate and prepare the best search model for molecular replacement. MrBUMP and BALBES are two recent developments from CCP4 that have been designed to automate and speed up the process of determining and preparing the best search models and putting them through molecular replacement. Their intention is to provide the user with a broad set of results using many search models and to highlight the best of these for further processing. An overview of both programs is presented along with a description of how best to use them, citing case studies and the results of large-scale testing of the software. text/html Evaluating the solution from MrBUMP and BALBES text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 313 research papers 0907-4449 med@iucr.org 323 http://scripts.iucr.org/cgi-bin/paper?ba5153 This paper presents a discussion of existing methods for the analysis of macromolecular interactions and complexes in crystal packing. Typical situations and conditions where wrong answers may be obtained in the course of ordinary procedures are presented and discussed. The more general question of what the relationship is between natural (in-solvent) and crystallized assemblies is discussed and researched. A computational analysis suggests that weak interactions with Kd ≥ 100 µM have a considerable chance of being lost during the course of crystallization. In such instances, crystal packing misrepresents macromolecular complexes and interactions. For as many as 20% of protein dimers in the PDB the likelihood of misrepresentation is estimated to be higher than 50%. Given that weak macromolecular interactions play an important role in many biochemical processes, these results suggest that a complementary noncrystallographic study should be always conducted when inferring structural aspects of weakly bound complexes. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Krissinel, E. 2011-04-01 doi:10.1107/S0907444911007232 International Union of Crystallography Methods for the analysis of the relationship between macromolecular complexes and interactions and their manifestation in crystal packing are described and discussed. en MACROMOLECULAR COMPLEXES; CRYSTAL PACKING; INTERACTIONS This paper presents a discussion of existing methods for the analysis of macromolecular interactions and complexes in crystal packing. Typical situations and conditions where wrong answers may be obtained in the course of ordinary procedures are presented and discussed. The more general question of what the relationship is between natural (in-solvent) and crystallized assemblies is discussed and researched. A computational analysis suggests that weak interactions with Kd ≥ 100 µM have a considerable chance of being lost during the course of crystallization. In such instances, crystal packing misrepresents macromolecular complexes and interactions. For as many as 20% of protein dimers in the PDB the likelihood of misrepresentation is estimated to be higher than 50%. Given that weak macromolecular interactions play an important role in many biochemical processes, these results suggest that a complementary noncrystallographic study should be always conducted when inferring structural aspects of weakly bound complexes. text/html Macromolecular complexes in crystals and solutions text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 376 research papers 0907-4449 med@iucr.org 385 http://scripts.iucr.org/cgi-bin/paper?ba5165 CCP4mg is a molecular-graphics program that is designed to give rapid access to both straightforward and complex static and dynamic representations of macromolecular structures. It has recently been updated with a new interface that provides more sophisticated atom-selection options and a wizard to facilitate the generation of complex scenes. These scenes may contain a mixture of coordinate-derived and abstract graphical objects, including text objects, arbitrary vectors, geometric objects and imported images, which can enhance a picture and eliminate the need for subsequent editing. Scene descriptions can be saved to file and transferred to other molecules. Here, the substantially enhanced version 2 of the program, with a new underlying GUI toolkit, is described. A built-in rendering module produces publication-quality images. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 McNicholas, S. Potterton, E. Wilson, K.S. Noble, M.E.M. 2011-04-01 doi:10.1107/S0907444911007281 International Union of Crystallography The CCP4 molecular-graphics program now uses the Qt framework to provide a modern look and feel. There are many new features including rendering for publication-quality images and sequence alignment. en CCP4MG; MOLECULAR GRAPHICS; MOVIES; CRYSTALLOGRAPHIC SOFTWARE CCP4mg is a molecular-graphics program that is designed to give rapid access to both straightforward and complex static and dynamic representations of macromolecular structures. It has recently been updated with a new interface that provides more sophisticated atom-selection options and a wizard to facilitate the generation of complex scenes. These scenes may contain a mixture of coordinate-derived and abstract graphical objects, including text objects, arbitrary vectors, geometric objects and imported images, which can enhance a picture and eliminate the need for subsequent editing. Scene descriptions can be saved to file and transferred to other molecules. Here, the substantially enhanced version 2 of the program, with a new underlying GUI toolkit, is described. A built-in rendering module produces publication-quality images. text/html Presenting your structures: the CCP4mg molecular-graphics software text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 386 research papers 0907-4449 med@iucr.org 394 http://scripts.iucr.org/cgi-bin/paper?ba5154 Density modification often suffers from an overestimation of phase quality, as seen by escalated figures of merit. A new cross-validation-based method to address this estimation bias by applying a bias-correction parameter `β' to maximum-likelihood phase-combination functions is proposed. In tests on over 100 single-wavelength anomalous diffraction data sets, the method is shown to produce much more reliable figures of merit and improved electron-density maps. Furthermore, significantly better results are obtained in automated model building iterated with phased refinement using the more accurate phase probability parameters from density modification. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Skubák, P. Pannu, N.S. 2011-04-01 doi:10.1107/S0907444911002083 International Union of Crystallography A cross-validation-based method for bias reduction in `classical' iterative density modification of experimental X-ray crystallography maps provides significantly more accurate phase-quality estimates and leads to improved automated model building. en RELIABLE FIGURE-OF-MERIT ESTIMATES; DENSITY MODIFICATION; MAXIMUM LIKELIHOOD; BIAS REDUCTION Density modification often suffers from an overestimation of phase quality, as seen by escalated figures of merit. A new cross-validation-based method to address this estimation bias by applying a bias-correction parameter `β' to maximum-likelihood phase-combination functions is proposed. In tests on over 100 single-wavelength anomalous diffraction data sets, the method is shown to produce much more reliable figures of merit and improved electron-density maps. Furthermore, significantly better results are obtained in automated model building iterated with phased refinement using the more accurate phase probability parameters from density modification. text/html Reduction of density-modification bias by β correction text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 345 research papers 0907-4449 med@iucr.org 354 http://scripts.iucr.org/cgi-bin/paper?ba5152 This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, `jelly-body' restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback–Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Murshudov, G.N. Skubák, P. Lebedev, A.A. Pannu, N.S. Steiner, R.A. Nicholls, R.A. Winn, M.D. Long, F. Vagin, A.A. 2011-04-01 doi:10.1107/S0907444911001314 International Union of Crystallography The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described. en REFMAC5; REFINEMENT This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, `jelly-body' restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback–Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography. text/html REFMAC5 for the refinement of macromolecular crystal structures text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 355 research papers 0907-4449 med@iucr.org 367 http://scripts.iucr.org/cgi-bin/paper?ba5157 For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single- or multiple-wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/). http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Pannu, N.S. Waterreus, W.-J. Skubák, P. Sikharulidze, I. Abrahams, J.P. de Graaff, R.A.G. 2011-04-01 doi:10.1107/S0907444910052224 International Union of Crystallography Recent developments in the CRANK software suite for experimental phasing have led to many more structures being built automatically. en CRANK; EXPERIMENTAL PHASING For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single- or multiple-wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/). text/html Recent advances in the CRANK software suite for experimental phasing text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 331 research papers 0907-4449 med@iucr.org 337 http://scripts.iucr.org/cgi-bin/paper?hm5093 E-ISA247 (voclosporin) is a cyclosporin A analogue that is in late-stage clinical development for the treatment of autoimmune diseases and the prevention of organ graft rejection. The X-­ray crystal structures of E-ISA247 and its stereoisomer Z-­ISA247 bound to cyclophilin A have been determined and their binding affinities were measured to be 15 and 61 nM, respectively, by fluorescence spectroscopy. The higher affinity of E-ISA247 can be explained by superior van der Waals contacts between its unique side chain and cyclophilin A. Comparison with the known ternary structure including calcineurin suggests that the higher immunosuppressive efficacy of E-­ISA247 relative to cyclosporin A could be a consequence of structural changes in calcineurin induced by the modified E-­ISA247 side chain. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kuglstatter, A. Mueller, F. Kusznir, E. Gsell, B. Stihle, M. Thoma, R. Benz, J. Aspeslet, L. Freitag, D. Hennig, M. 2011-02-01 doi:10.1107/S0907444910051905 International Union of Crystallography X-ray crystal structures of the cyclosporin A analogue E-ISA247 (voclosporin) and its stereoisomer Z-ISA247 bound to cyclophilin A suggest the molecular basis for the differences in their binding affinities and immunosuppressive efficacies. en VOCLOSPORIN; CYCLOPHILIN A; E-ISA247; Z-ISA247 E-ISA247 (voclosporin) is a cyclosporin A analogue that is in late-stage clinical development for the treatment of autoimmune diseases and the prevention of organ graft rejection. The X-­ray crystal structures of E-ISA247 and its stereoisomer Z-­ISA247 bound to cyclophilin A have been determined and their binding affinities were measured to be 15 and 61 nM, respectively, by fluorescence spectroscopy. The higher affinity of E-ISA247 can be explained by superior van der Waals contacts between its unique side chain and cyclophilin A. Comparison with the known ternary structure including calcineurin suggests that the higher immunosuppressive efficacy of E-­ISA247 relative to cyclosporin A could be a consequence of structural changes in calcineurin induced by the modified E-­ISA247 side chain. text/html Structural basis for the cyclophilin A binding affinity and immunosuppressive potency of E-­ISA247 (voclosporin) text 67 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-02-01 119 research papers 0907-4449 med@iucr.org 123 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5159 Phaser is a program that implements likelihood-based methods to solve macromolecular crystal structures, currently by molecular replacement or single-wavelength anomalous diffraction (SAD). SAD phasing is based on a likelihood target derived from the joint probability distribution of observed and calculated pairs of Friedel-related structure factors. This target combines information from the total structure factor (primarily non-anomalous scattering) and the difference between the Friedel mates (anomalous scattering). Phasing starts from a substructure, which is usually but not necessarily a set of anomalous scatterers. The substructure can also be a protein model, such as one obtained by molecular replacement. Additional atoms are found using a log-likelihood gradient map, which shows the sites where the addition of scattering from a particular atom type would improve the likelihood score. An automated completion algorithm adds new sites, choosing optionally among different atom types, adds anisotropic B-factor parameters if appropriate and deletes atoms that refine to low occupancy. Log-likelihood gradient maps can also identify which atoms in a refined protein structure are anomalous scatterers, such as metal or halide ions. These maps are more sensitive than conventional model-phased anomalous difference Fouriers and the iterative completion algorithm is able to find a significantly larger number of convincing sites. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Read, R.J. McCoy, A.J. 2011-04-01 doi:10.1107/S0907444910051371 International Union of Crystallography SAD data can be used in Phaser to solve novel structures, supplement molecular-replacement phase information or identify anomalous scatterers from a final refined model. en SAD PHASING; LIKELIHOOD; MOLECULAR REPLACEMENT Phaser is a program that implements likelihood-based methods to solve macromolecular crystal structures, currently by molecular replacement or single-wavelength anomalous diffraction (SAD). SAD phasing is based on a likelihood target derived from the joint probability distribution of observed and calculated pairs of Friedel-related structure factors. This target combines information from the total structure factor (primarily non-anomalous scattering) and the difference between the Friedel mates (anomalous scattering). Phasing starts from a substructure, which is usually but not necessarily a set of anomalous scatterers. The substructure can also be a protein model, such as one obtained by molecular replacement. Additional atoms are found using a log-likelihood gradient map, which shows the sites where the addition of scattering from a particular atom type would improve the likelihood score. An automated completion algorithm adds new sites, choosing optionally among different atom types, adds anisotropic B-factor parameters if appropriate and deletes atoms that refine to low occupancy. Log-likelihood gradient maps can also identify which atoms in a refined protein structure are anomalous scatterers, such as metal or halide ions. These maps are more sensitive than conventional model-phased anomalous difference Fouriers and the iterative completion algorithm is able to find a significantly larger number of convincing sites. text/html Using SAD data in Phaser text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 338 research papers 0907-4449 med@iucr.org 344 http://scripts.iucr.org/cgi-bin/paper?ba5163 In molecular replacement, the quality of models can be improved by transferring information contained in sequence alignment to the template structure. A family of algorithms has been developed that make use of the sequence-similarity score calculated from residue-substitution scores smoothed over nearby residues to delete or downweight parts of the model that are unreliable. These algorithms have been implemented in the program Sculptor, together with well established methods that are in common use for model improvement. An analysis of the new algorithms has been performed by studying the effect of algorithm parameters on the quality of models. Benchmarking against existing tech­niques shows that models from Sculptor compare favourably, especially if the alignment is unreliable. Carrying out multiple trials using alternative models created from the same structure but using different algorithm parameters can significantly improve the success rate. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Bunkóczi, G. Read, R.J. 2011-04-01 doi:10.1107/S0907444910051218 International Union of Crystallography The molecular-replacement model-improvement program Sculptor is described, with an analysis of the algorithms used. en MOLECULAR REPLACEMENT; MODEL IMPROVEMENT; RESIDUE-SUBSTITUTION SCORE In molecular replacement, the quality of models can be improved by transferring information contained in sequence alignment to the template structure. A family of algorithms has been developed that make use of the sequence-similarity score calculated from residue-substitution scores smoothed over nearby residues to delete or downweight parts of the model that are unreliable. These algorithms have been implemented in the program Sculptor, together with well established methods that are in common use for model improvement. An analysis of the new algorithms has been performed by studying the effect of algorithm parameters on the quality of models. Benchmarking against existing tech­niques shows that models from Sculptor compare favourably, especially if the alignment is unreliable. Carrying out multiple trials using alternative models created from the same structure but using different algorithm parameters can significantly improve the success rate. text/html Improvement of molecular-replacement models with Sculptor text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 303 research papers 0907-4449 med@iucr.org 312 http://scripts.iucr.org/cgi-bin/paper?ba5160 iMOSFLM is a graphical user interface to the diffraction data-integration program MOSFLM. It is designed to simplify data processing by dividing the process into a series of steps, which are normally carried out sequentially. Each step has its own display pane, allowing control over parameters that influence that step and providing graphical feedback to the user. Suitable values for integration parameters are set automatically, but additional menus provide a detailed level of control for experienced users. The image display and the interfaces to the different tasks (indexing, strategy calculation, cell refinement, integration and history) are described. The most important parameters for each step and the best way of assessing success or failure are discussed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Battye, T.G.G. Kontogiannis, L. Johnson, O. Powell, H.R. Leslie, A.G.W. 2011-04-01 doi:10.1107/S0907444910048675 International Union of Crystallography A new graphical user interface to the MOSFLM program has been developed to simplify the processing of macromolecular diffraction data. The interface, iMOSFLM, allows data processing via a series of clearly defined tasks and provides visual feedback on the progress of each stage. en DATA INTEGRATION; GRAPHICAL USER INTERFACE; DATA PROCESSING; SOFTWARE iMOSFLM is a graphical user interface to the diffraction data-integration program MOSFLM. It is designed to simplify data processing by dividing the process into a series of steps, which are normally carried out sequentially. Each step has its own display pane, allowing control over parameters that influence that step and providing graphical feedback to the user. Suitable values for integration parameters are set automatically, but additional menus provide a detailed level of control for experienced users. The image display and the interfaces to the different tasks (indexing, strategy calculation, cell refinement, integration and history) are described. The most important parameters for each step and the best way of assessing success or failure are discussed. text/html iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 271 research papers 0907-4449 med@iucr.org 281 http://scripts.iucr.org/cgi-bin/paper?dz5219 The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallo­graphy is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Winn, M.D. Ballard, C.C. Cowtan, K.D. Dodson, E.J. Emsley, P. Evans, P.R. Keegan, R.M. Krissinel, E.B. Leslie, A.G.W. McCoy, A. McNicholas, S.J. Murshudov, G.N. Pannu, N.S. Potterton, E.A. Powell, H.R. Read, R.J. Vagin, A. Wilson, K.S. 2011-04-01 doi:10.1107/S0907444910045749 International Union of Crystallography An overview of the CCP4 software suite for macromolecular crystallography is given. en CCP4; MACROMOLECULAR CRYSTALLOGRAPHY; SOFTWARE; COLLABORATION; AUTOMATION; MACROMOLECULAR STRUCTURE DETERMINATION The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallo­graphy is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package. text/html Overview of the CCP4 suite and current developments text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 235 research papers 0907-4449 med@iucr.org 242 http://scripts.iucr.org/cgi-bin/paper?me0429 Copyright (c) 2010 International Union of Crystallography urn:issn:0907-4449 Weiss, M.S. Einspahr, H. Baker, E.N. Dauter, Z. Kaysser-Pyzalla Kaysser-Pyzalla, A. Kostorz, G. Larsen, S. 2010-12-01 doi:10.1107/S0907444910041818 International Union of Crystallography The problem of undercounting of citations that are published only in supplementary material is studied for the journals Nature, Science, Cell and the Proceedings of the National Academy of Sciences (USA). en CITATIONS; SUPPLEMENTARY MATERIAL; EDITORIAL text/html Citations in supplementary material text 66 12 Copyright (c) 2010 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2010-12-01 1269 editorial 0907-4449 med@iucr.org 1270 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5155 The Protein Data Bank in Europe (PDBe) is the European partner in the Worldwide PDB and as such handles depositions of X-ray, NMR and EM data and structure models. PDBe also provides advanced bioinformatics services based on data from the PDB and related resources. Some of the challenges facing the PDB and its guardians are discussed, as well as some of the areas on which PDBe activities will focus in the future (advanced services, ligands, integration, validation and experimental data). Finally, some recent developments at PDBe are described. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Velankar, S. Kleywegt, G.J. 2011-04-01 doi:10.1107/S090744491004117X International Union of Crystallography Some future challenges for the PDB and its guardians are discussed and current and future activities in structural bioinformatics at the Protein Data Bank in Europe (PDBe) are described. en PROTEIN DATA BANK IN EUROPE The Protein Data Bank in Europe (PDBe) is the European partner in the Worldwide PDB and as such handles depositions of X-ray, NMR and EM data and structure models. PDBe also provides advanced bioinformatics services based on data from the PDB and related resources. Some of the challenges facing the PDB and its guardians are discussed, as well as some of the areas on which PDBe activities will focus in the future (advanced services, ligands, integration, validation and experimental data). Finally, some recent developments at PDBe are described. text/html The Protein Data Bank in Europe (PDBe): bringing structure to biology text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 324 research papers 0907-4449 med@iucr.org 330 http://scripts.iucr.org/cgi-bin/paper?kw5028 The Engh and Huber parameters for bond lengths and bond angles have been used uncontested in macromolecular structure refinement from 1991 until very recently, despite critical discussion of their ubiquitous validity by many authors. An extensive analysis of the backbone angle τ (N—Cα—C) illustrates that the Engh and Huber parameters can indeed be improved and a recent study [Tronrud et al. (2010), Acta Cryst. D66, 834–842] confirms these ideas. However, the present study of τ shows that improving the Engh and Huber parameters will be considerably more complex than simply making the parameters a function of the backbone ϕ, ψ angles. Many other aspects, such as the cooperativity of hydrogen bonds, the bending of secondary-structure elements and a series of biophysical aspects of the 20 amino-acid types, will also need to be taken into account. Different sets of Engh and Huber parameters will be needed for conceptually different refinement programs. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Touw, W.G. Vriend, G. 2010-12-01 doi:10.1107/S0907444910040928 International Union of Crystallography The angle τ (backbone N—Cα—C) is the most contested Engh and Huber refinement target parameter. It is shown that this parameter is `correct' as a PDB-wide average, but can be improved by taking into account residue types, secondary structures and many other aspects of our knowledge of the biophysical relations between residue type and protein structure. en PROTEIN STRUCTURE; PROTEIN GEOMETRY; STEREOCHEMICAL PARAMETERS; RESTRAINTS; N-C[ALPHA]-C BOND ANGLE The Engh and Huber parameters for bond lengths and bond angles have been used uncontested in macromolecular structure refinement from 1991 until very recently, despite critical discussion of their ubiquitous validity by many authors. An extensive analysis of the backbone angle τ (N—Cα—C) illustrates that the Engh and Huber parameters can indeed be improved and a recent study [Tronrud et al. (2010), Acta Cryst. D66, 834–842] confirms these ideas. However, the present study of τ shows that improving the Engh and Huber parameters will be considerably more complex than simply making the parameters a function of the backbone ϕ, ψ angles. Many other aspects, such as the cooperativity of hydrogen bonds, the bending of secondary-structure elements and a series of biophysical aspects of the 20 amino-acid types, will also need to be taken into account. Different sets of Engh and Huber parameters will be needed for conceptually different refinement programs. text/html On the complexity of Engh and Huber refinement restraints: the angle τ as example text 66 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-12-01 1341 research papers 0907-4449 med@iucr.org 1350 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5156 The Joint Center for Structural Genomics (JCSG), one of four large-scale structure-determination centers funded by the US Protein Structure Initiative (PSI) through the National Institute for General Medical Sciences, has been operating an automated distributed structure-solution pipeline, Xsolve, for well over half a decade. During PSI-2, Xsolve solved, traced and partially refined 90% of the JCSG's nearly 770 MAD/SAD structures at an average resolution of about 2 Å without human intervention. Xsolve executes many well established publicly available crystallography software programs in parallel on a commodity Linux cluster, resulting in multiple traces for any given target. Additional software programs have been developed and integrated into Xsolve to further minimize human effort in structure refinement. ConsensusModeler exploits complementarities in traces from Xsolve to compute a single optimal model for manual refinement. Xpleo is a powerful robotics-inspired algorithm to build missing fragments and qFit automatically identifies and fits alternate conformations. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 van den Bedem, H. Wolf, G. Xu, Q. Deacon, A.M. 2011-04-01 doi:10.1107/S0907444910039934 International Union of Crystallography The software suite Xsolve semi-exhaustively explores key parameters of the X-ray structure-determination process to compute multiple three-dimensional protein structures independently and in parallel from a set of diffraction images. An optimal consensus model for subsequent manual refinement is computed from these structures. en DISTRIBUTED PROTEIN-STRUCTURE DETERMINATION; CONSENSUS MODELS; PARALLEL COMPUTING The Joint Center for Structural Genomics (JCSG), one of four large-scale structure-determination centers funded by the US Protein Structure Initiative (PSI) through the National Institute for General Medical Sciences, has been operating an automated distributed structure-solution pipeline, Xsolve, for well over half a decade. During PSI-2, Xsolve solved, traced and partially refined 90% of the JCSG's nearly 770 MAD/SAD structures at an average resolution of about 2 Å without human intervention. Xsolve executes many well established publicly available crystallography software programs in parallel on a commodity Linux cluster, resulting in multiple traces for any given target. Additional software programs have been developed and integrated into Xsolve to further minimize human effort in structure refinement. ConsensusModeler exploits complementarities in traces from Xsolve to compute a single optimal model for manual refinement. Xpleo is a powerful robotics-inspired algorithm to build missing fragments and qFit automatically identifies and fits alternate conformations. text/html Distributed structure determination at the JCSG text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 368 research papers 0907-4449 med@iucr.org 375 http://scripts.iucr.org/cgi-bin/paper?ba5158 This paper presents an overview of how to run the CCP4 programs for data reduction (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers determination of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examination of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estimation of |F| from intensity and finally examination of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Evans, P.R. 2011-04-01 doi:10.1107/S090744491003982X International Union of Crystallography A summary of how to run the data-reduction programs in the CCP4 suite. en CCP4; DATA REDUCTION; DATA SCALING; SOFTWARE This paper presents an overview of how to run the CCP4 programs for data reduction (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers determination of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examination of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estimation of |F| from intensity and finally examination of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups. text/html An introduction to data reduction: space-group determination, scaling and intensity statistics text 67 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2011-04-01 282 research papers 0907-4449 med@iucr.org 292 http://scripts.iucr.org/cgi-bin/paper?me0425 Copyright (c) 2010 International Union of Crystallography urn:issn:0907-4449 Adams, P. Langan, P. 2010-11-01 doi:10.1107/S0907444910039387 International Union of Crystallography A summary of the current state of neutron crystallography is given. en EDITORIAL; NEUTRONS; NEUTRON CRYSTALLOGRAPHY text/html Opportunities and challenges with the growth of neutron crystallography text 66 11 Copyright (c) 2010 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2010-11-01 1121 editorial 0907-4449 med@iucr.org 1123 http://scripts.iucr.org/cgi-bin/paper?mn5003 Inclusion of low-resolution data in macromolecular crystallo­graphy requires a model for the bulk solvent. Previous methods have used a binary mask to accomplish this, which has proven to be very effective, but the mask is discontinuous at the solute–solvent boundary (i.e. the mask value jumps from zero to one) and is not differentiable with respect to atomic parameters. Here, two algorithms are introduced for com­puting bulk-solvent models using either a polynomial switch or a smoothly thresholded product of Gaussians, and both models are shown to be efficient and differentiable with respect to atomic coordinates. These alternative bulk-solvent models offer algorithmic improvements, while showing similar agreement of the model with the observed amplitudes relative to the binary model as monitored using R, Rfree and differences between experimental and model phases. As with the standard solvent models, the alternative models improve the agreement primarily with lower resolution (>6 Å) data versus no bulk solvent. The models are easily implemented into crystallographic software packages and can be used as a general method for bulk-solvent correction in macromolecular crystallography. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Fenn, T.D. Schnieders, M.J. Brunger, A.T. 2010-09-01 doi:10.1107/S0907444910031045 International Union of Crystallography A new method for modeling the bulk solvent in macromolecular diffraction data based on Babinet's principle is presented. The proposed models offer the advantage of differentiability with respect to atomic coordinates. en BULK-SOLVENT MODELS Inclusion of low-resolution data in macromolecular crystallo­graphy requires a model for the bulk solvent. Previous methods have used a binary mask to accomplish this, which has proven to be very effective, but the mask is discontinuous at the solute–solvent boundary (i.e. the mask value jumps from zero to one) and is not differentiable with respect to atomic parameters. Here, two algorithms are introduced for com­puting bulk-solvent models using either a polynomial switch or a smoothly thresholded product of Gaussians, and both models are shown to be efficient and differentiable with respect to atomic coordinates. These alternative bulk-solvent models offer algorithmic improvements, while showing similar agreement of the model with the observed amplitudes relative to the binary model as monitored using R, Rfree and differences between experimental and model phases. As with the standard solvent models, the alternative models improve the agreement primarily with lower resolution (>6 Å) data versus no bulk solvent. The models are easily implemented into crystallographic software packages and can be used as a general method for bulk-solvent correction in macromolecular crystallography. text/html A smooth and differentiable bulk-solvent model for macromolecular diffraction text 66 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-09-01 1024 research papers 0907-4449 med@iucr.org 1031 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5161 In Escherichia coli and many other bacterial species, the glycolytic enzyme enolase is a component of the multi-enzyme RNA degradosome, an assembly that is involved in RNA processing and degradation. Enolase is recruited into the degradosome through interactions with a small recognition motif located within the degradosome-scaffolding domain of RNase E. Here, the crystal structure of enolase bound to its cognate site from RNase E (residues 823–850) at 1.9 Å resolution is presented. The structure suggests that enolase may help to organize an adjacent conserved RNA-binding motif in RNase E. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Nurmohamed, S. McKay, A.R. Robinson, C.V. Luisi, B.F. 2010-09-01 doi:10.1107/S0907444910030015 International Union of Crystallography The glycolytic enzyme enolase associates with the endoribonuclease RNase E in Escherichia coli and many other bacterial species. The crystal structure of the complex reveals the basis for the molecular recognition and provides clues as to the possible function of the interaction. en ENOLASE; RNA DEGRADOSOME; RNA PROCESSING; NATIVELY UNFOLDED PROTEINS In Escherichia coli and many other bacterial species, the glycolytic enzyme enolase is a component of the multi-enzyme RNA degradosome, an assembly that is involved in RNA processing and degradation. Enolase is recruited into the degradosome through interactions with a small recognition motif located within the degradosome-scaffolding domain of RNase E. Here, the crystal structure of enolase bound to its cognate site from RNase E (residues 823–850) at 1.9 Å resolution is presented. The structure suggests that enolase may help to organize an adjacent conserved RNA-binding motif in RNase E. text/html Molecular recognition between Escherichia coli enolase and ribonuclease E text 66 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-09-01 1036 short communications 0907-4449 med@iucr.org 1040 1399-0047 http://scripts.iucr.org/cgi-bin/paper?en5421 The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to Rcryst = 23.7% (Rfree = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Morgunova, E. Illarionov, B. Saller, S. Popov, A. Sambaiah, T. Bacher, A. Cushman, M. Fischer, M. Ladenstein, R. 2010-09-01 doi:10.1107/S0907444910029690 International Union of Crystallography Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. en BACILLUS ANTHRACIS; RIBOFLAVIN BIOSYNTHESIS; LUMAZINE SYNTHASE; ANTHRAX; INHIBITION; DRUG DESIGN The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to Rcryst = 23.7% (Rfree = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis. text/html Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis text 66 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-09-01 1001 research papers 0907-4449 med@iucr.org 1011 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5150 While figures of speech are often useful and even educational, flashy titles combined with hyperbole and imprecise language can mislead or deceive non-specialist readers and should therefore be avoided. The possibility of such confusion exists when poorly defined terms like `structure quality' or `super-resolution' are used to describe a protein structure. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Wlodawer, A. Lubkowski, J. Minor, W. Jaskolski. 2010-09-01 doi:10.1107/S090744491002799X International Union of Crystallography A brief comment is made on the need to use carefully selected, novel terms in crystallographic publications, especially publications addressing non-specialists. en LETTERS TO THE EDITOR; CRYSTALLOGRAPHIC TERMINOLOGY While figures of speech are often useful and even educational, flashy titles combined with hyperbole and imprecise language can mislead or deceive non-specialist readers and should therefore be avoided. The possibility of such confusion exists when poorly defined terms like `structure quality' or `super-resolution' are used to describe a protein structure. text/html Is too `creative' language acceptable in crystallography? text 66 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-09-01 1041 letters to the editor 0907-4449 med@iucr.org 1042 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5361 The application of microseed matrix screening to the crystallization of antibody–antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Obmolova, G. Malia, T.J. Teplyakov, A. Sweet, R. Gilliland, G.L. 2010-08-01 doi:10.1107/S0907444910026041 International Union of Crystallography The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. en CRYSTALLIZATION; MICROSEED MATRIX SCREENING; ANTIBODY-ANTIGEN COMPLEXES The application of microseed matrix screening to the crystallization of antibody–antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. text/html Promoting crystallization of antibody–antigen complexes via microseed matrix screening text 66 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-08-01 927 research papers 0907-4449 med@iucr.org 933 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5035 The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit–subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Fyfe, P.K. Dawson, A. Hutchison, M.-T. Cameron, S. Hunter, W.N. 2010-08-01 doi:10.1107/S0907444910020081 International Union of Crystallography The 2.5 Å resolution structure of S. aureus adenylosuccinate lyase is reported and compared with those of orthologues to assess its potential as a template for early stage drug discovery. AMP and a putative assignment of oxalate, the latter an artefact possibly arising from an impurity in the PEG used for crystallization, occupy the active site. en ADENYLOSUCCINATE LYASE; AMP; OXALATE; PURINE BIOSYNTHESIS; PURINE CYCLE The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit–subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization. text/html Structure of Staphylococcus aureus adenylo­succinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discovery text 66 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-08-01 881 research papers 0907-4449 med@iucr.org 888 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5152 Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are con­served across species. Arabidopsis thaliana monothiol gluta­redoxin AtGRXcp is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been determined at 2.4 Å resolution. AtGRXcp has a glutaredoxin/thioredoxin-like fold with distinct structural features that differ from those of dithiol Grxs. The structure reveals that the putative active-site motif CGFS is well defined and is located on the molecular surface and that a long groove extends to both sides of the catalytic Cys97. Structural comparison and molecular modeling suggest that glutathione can bind in this groove and form extensive interactions with conserved charged residues including Lys89, Arg126 and Asp152. Further comparative studies reveal that a unique loop with five additional residues adjacent to the active-site motif may be a key structural feature of monothiol Grxs and may influence their function. This study provides the first structural information on plant CGFS-type monothiol Grxs, allowing a better understanding of the redox-regulation mechanism mediated by these plant Grxs. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Li, L. Cheng, N. Hirschi, K.D. Wang, X. 2010-06-01 doi:10.1107/S0907444910013119 International Union of Crystallography The structure of Arabidopsis monothiol glutaredoxin AtGRXcp has been determined and reveals unique structural features of monothiol glutaredoxins, key residues for their interaction with glutathione and structural determinants for their distinct biochemical properties. en MONOTHIOL GLUTAREDOXINS; ATGRXCP; ARABIDOPSIS THALIANA Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are con­served across species. Arabidopsis thaliana monothiol gluta­redoxin AtGRXcp is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been determined at 2.4 Å resolution. AtGRXcp has a glutaredoxin/thioredoxin-like fold with distinct structural features that differ from those of dithiol Grxs. The structure reveals that the putative active-site motif CGFS is well defined and is located on the molecular surface and that a long groove extends to both sides of the catalytic Cys97. Structural comparison and molecular modeling suggest that glutathione can bind in this groove and form extensive interactions with conserved charged residues including Lys89, Arg126 and Asp152. Further comparative studies reveal that a unique loop with five additional residues adjacent to the active-site motif may be a key structural feature of monothiol Grxs and may influence their function. This study provides the first structural information on plant CGFS-type monothiol Grxs, allowing a better understanding of the redox-regulation mechanism mediated by these plant Grxs. text/html Structure of Arabidopsis chloroplastic monothiol glutaredoxin AtGRXcp text 66 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-06-01 725 research papers 0907-4449 med@iucr.org 732 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ea5120 Protein type III secretion systems (T3SSs) are organic nanosyringes that achieve an energy-dependent translocation of bacterial proteins through the two membranes of Gram-negative organisms. Examples include the pathogenic systems of animals, plants and symbiotic bacteria that inject factors into eukaryotic cells, and the flagellar export system that secretes flagellin. T3SSs possess a core of several membrane-associated proteins that are conserved across all known bacterial species that use this system. The Salmonella protein InvA is one of the most highly conserved proteins of this core of critical T3SS components. The crystal structure of a C-­terminal domain of InvA reveals an unexpected homology to domains that have been repeatedly found as building blocks of other elements of the T3SS apparatus. This suggests the surprising hypothesis that evolution has produced a significant component of the apparatus structure through a series of gene-duplication and gene-rearrangement events. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Lilic, M. Quezada, C.M. Stebbins, C.E. 2010-06-01 doi:10.1107/S0907444910010796 International Union of Crystallography The cytoplasmic domain of Salmonella InvA shares homology to a recurring scaffold in the membrane-spanning components of the type II and type III secretion systems. en SALMONELLA; TYPE III SECRETION; BACTERIAL PATHOGENESIS; PROTEIN TRANSLOCATION Protein type III secretion systems (T3SSs) are organic nanosyringes that achieve an energy-dependent translocation of bacterial proteins through the two membranes of Gram-negative organisms. Examples include the pathogenic systems of animals, plants and symbiotic bacteria that inject factors into eukaryotic cells, and the flagellar export system that secretes flagellin. T3SSs possess a core of several membrane-associated proteins that are conserved across all known bacterial species that use this system. The Salmonella protein InvA is one of the most highly conserved proteins of this core of critical T3SS components. The crystal structure of a C-­terminal domain of InvA reveals an unexpected homology to domains that have been repeatedly found as building blocks of other elements of the T3SS apparatus. This suggests the surprising hypothesis that evolution has produced a significant component of the apparatus structure through a series of gene-duplication and gene-rearrangement events. text/html A conserved domain in type III secretion links the cytoplasmic domain of InvA to elements of the basal body text 66 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-06-01 709 research papers 0907-4449 med@iucr.org 713 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5150 Radiation damage inflicted during diffraction data collection in macromolecular crystallography has re-emerged in the last decade as a major experimental and computational challenge, as even for crystals held at 100 K it can result in severe data-quality degradation and the appearance in solved structures of artefacts which affect biological interpretations. Here, the observable symptoms and basic physical processes involved in radiation damage are described and the concept of absorbed dose as the basic metric against which to monitor the experimentally observed changes is outlined. Investigations into radiation damage in macromolecular crystallography are ongoing and the number of studies is rapidly increasing. The current literature on the subject is compiled as a resource for the interested researcher. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Garman, E.F. 2010-04-01 doi:10.1107/S0907444910008656 International Union of Crystallography The basic causes of the radiation damage inflicted on macromolecular crystals during diffraction experiments are summarized, as well as the current state of research which attempts to understand and to mitigate it. en RADIATION DAMAGE; CRYOCRYSTALLOGRAPHY; ROOM-TEMPERATURE MACROMOLECULAR CRYSTALLOGRAPHY; DOSE Radiation damage inflicted during diffraction data collection in macromolecular crystallography has re-emerged in the last decade as a major experimental and computational challenge, as even for crystals held at 100 K it can result in severe data-quality degradation and the appearance in solved structures of artefacts which affect biological interpretations. Here, the observable symptoms and basic physical processes involved in radiation damage are described and the concept of absorbed dose as the basic metric against which to monitor the experimentally observed changes is outlined. Investigations into radiation damage in macromolecular crystallography are ongoing and the number of studies is rapidly increasing. The current literature on the subject is compiled as a resource for the interested researcher. text/html Radiation damage in macromolecular crystallography: what is it and why should we care? text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 339 research papers 0907-4449 med@iucr.org 351 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5144 Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are `discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Emsley, P. Lohkamp, B. Scott, W.G. Cowtan, K. 2010-04-01 doi:10.1107/S0907444910007493 International Union of Crystallography Coot is a molecular-graphics program designed to assist in the building of protein and other macromolecular models. The current state of development and available features are presented. en COOT; MODEL BUILDING Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are `discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed. text/html Features and development of Coot text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 486 research papers 0907-4449 med@iucr.org 501 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5148 In this work, classic intensity formulae were united with an empirical spot-fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per spot at a desired resolution over the radiation-damage-limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X-ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal-to-noise ratio of 2 at 2 Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2 µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34 µm. These represent 15-fold to 700-fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Holton, J.M. Frankel, K.A. 2010-04-01 doi:10.1107/S0907444910007262 International Union of Crystallography A formula for absolute scattering power is derived to include spot fading arising from radiation damage and the crystal volume needed to collect diffraction data to a given resolution is calculated. en RADIATION DAMAGE; MINIMUM CRYSTAL SIZE; PROTEIN MACROMOLECULAR CRYSTALLOGRAPHY; SCATTERING POWER In this work, classic intensity formulae were united with an empirical spot-fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per spot at a desired resolution over the radiation-damage-limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X-ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal-to-noise ratio of 2 at 2 Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2 µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34 µm. These represent 15-fold to 700-fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits. text/html The minimum crystal size needed for a complete diffraction data set text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 393 research papers 0907-4449 med@iucr.org 408 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5146 The program RADDOSE is widely used to compute the dose absorbed by a macromolecular crystal during an X-ray diffraction experiment. A number of factors affect the absorbed dose, including the incident X-ray flux density, the photon energy and the composition of the macromolecule and of the buffer in the crystal. An experimental dose limit for macromolecular crystallography (MX) of 30 MGy at 100 K has been reported, beyond which the biological information obtained may be compromised. Thus, for the planning of an optimized diffraction experiment the estimation of dose has become an additional tool. A number of approximations were made in the original version of RADDOSE. Recently, the code has been modified in order to take into account fluorescent X-­ray escape from the crystal (version 2) and the inclusion of incoherent (Compton) scattering into the dose calculation is now reported (version 3). The Compton cross-section, although negligible at the energies currently commonly used in MX, should be considered in dose calculations for incident energies above 20 keV. Calculations using version 3 of RADDOSE reinforce previous studies that predict a reduction in the absorbed dose when data are collected at higher energies compared with data collected at 12.4 keV. Hence, a longer irradiation lifetime for the sample can be achieved at these higher energies but this is at the cost of lower diffraction intensities. The parameter `diffraction-dose efficiency', which is the diffracted intensity per absorbed dose, is revisited in an attempt to investigate the benefits and pitfalls of data collection using higher and lower energy radiation, particularly for thin crystals. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Paithankar, K.S. Garman, E.F. 2010-04-01 doi:10.1107/S0907444910006724 International Union of Crystallography The program RADDOSE computes the dose absorbed by a macromolecular crystal and here a guide is provided to help to ensure the proper use of the program. In the new version (v.3) described here, modifications to include the energy deposited owing to Compton scattering have been made. en RADIATION DAMAGE; ABSORBED DOSE; COMPTON SCATTERING; X-RAY FLUORESCENT ESCAPE; COMPTON ELECTRONS; MACROMOLECULAR CRYSTALLOGRAPHY; RADDOSE The program RADDOSE is widely used to compute the dose absorbed by a macromolecular crystal during an X-ray diffraction experiment. A number of factors affect the absorbed dose, including the incident X-ray flux density, the photon energy and the composition of the macromolecule and of the buffer in the crystal. An experimental dose limit for macromolecular crystallography (MX) of 30 MGy at 100 K has been reported, beyond which the biological information obtained may be compromised. Thus, for the planning of an optimized diffraction experiment the estimation of dose has become an additional tool. A number of approximations were made in the original version of RADDOSE. Recently, the code has been modified in order to take into account fluorescent X-­ray escape from the crystal (version 2) and the inclusion of incoherent (Compton) scattering into the dose calculation is now reported (version 3). The Compton cross-section, although negligible at the energies currently commonly used in MX, should be considered in dose calculations for incident energies above 20 keV. Calculations using version 3 of RADDOSE reinforce previous studies that predict a reduction in the absorbed dose when data are collected at higher energies compared with data collected at 12.4 keV. Hence, a longer irradiation lifetime for the sample can be achieved at these higher energies but this is at the cost of lower diffraction intensities. The parameter `diffraction-dose efficiency', which is the diffracted intensity per absorbed dose, is revisited in an attempt to investigate the benefits and pitfalls of data collection using higher and lower energy radiation, particularly for thin crystals. text/html Know your dose: RADDOSE text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 381 research papers 0907-4449 med@iucr.org 388 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5147 When collecting X-ray diffraction data from a crystal, we measure the intensities of the diffracted waves scattered from a series of planes that we can imagine slicing through the crystal in all directions. From these intensities we derive the amplitudes of the scattered waves, but in the experiment we lose the phase information; that is, how we offset these waves when we add them together to reconstruct an image of our molecule. This is generally known as the `phase problem'. We can only derive the phases from some knowledge of the molecular structure. In small-molecule crystallography, some basic assumptions about atomicity give rise to relationships between the amplitudes from which phase information can be extracted. In protein crystallography, these ab initio methods can only be used in the rare cases in which there are data to at least 1.2 Å resolution. For the majority of cases in protein crystallography phases are derived either by using the atomic coordinates of a structurally similar protein (molecular replacement) or by finding the positions of heavy atoms that are intrinsic to the protein or that have been added (methods such as MIR, MIRAS, SIR, SIRAS, MAD, SAD or com­binations of these). The pioneering work of Perutz, Kendrew, Blow, Crick and others developed the methods of isomorphous replacement: adding electron-dense atoms to the protein without disturbing the protein structure. Nowadays, methods from small-molecule crystallography can be used to find the heavy-atom substructure and the phases for the whole protein can be bootstrapped from this prior knowledge. More recently, improved X-ray sources, detectors and software have led to the routine use of anomalous scattering to obtain phase information from either incorporated selenium or intrinsic sulfurs. In the best cases, only a single set of X-ray data (SAD) is required to provide the positions of the anomalous scatters, which together with density-modification procedures can reveal the structure of the complete protein. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Taylor, G.L. 2010-04-01 doi:10.1107/S0907444910006694 International Union of Crystallography This introductory paper to the CCP4 weekend on experimental phasing introduces the concept of the `phase problem' for non-experts. Modern methods of phasing are explored, including some recent examples that can be downloaded as tutorials. en PHASING When collecting X-ray diffraction data from a crystal, we measure the intensities of the diffracted waves scattered from a series of planes that we can imagine slicing through the crystal in all directions. From these intensities we derive the amplitudes of the scattered waves, but in the experiment we lose the phase information; that is, how we offset these waves when we add them together to reconstruct an image of our molecule. This is generally known as the `phase problem'. We can only derive the phases from some knowledge of the molecular structure. In small-molecule crystallography, some basic assumptions about atomicity give rise to relationships between the amplitudes from which phase information can be extracted. In protein crystallography, these ab initio methods can only be used in the rare cases in which there are data to at least 1.2 Å resolution. For the majority of cases in protein crystallography phases are derived either by using the atomic coordinates of a structurally similar protein (molecular replacement) or by finding the positions of heavy atoms that are intrinsic to the protein or that have been added (methods such as MIR, MIRAS, SIR, SIRAS, MAD, SAD or com­binations of these). The pioneering work of Perutz, Kendrew, Blow, Crick and others developed the methods of isomorphous replacement: adding electron-dense atoms to the protein without disturbing the protein structure. Nowadays, methods from small-molecule crystallography can be used to find the heavy-atom substructure and the phases for the whole protein can be bootstrapped from this prior knowledge. More recently, improved X-ray sources, detectors and software have led to the routine use of anomalous scattering to obtain phase information from either incorporated selenium or intrinsic sulfurs. In the best cases, only a single set of X-ray data (SAD) is required to provide the positions of the anomalous scatters, which together with density-modification procedures can reveal the structure of the complete protein. text/html Introduction to phasing text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 325 research papers 0907-4449 med@iucr.org 338 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5142 Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces com­bined real–imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the software Phaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 McCoy, A.J. Read, R.J. 2010-04-01 doi:10.1107/S0907444910006335 International Union of Crystallography The pitfalls of experimental phasing are described. en ENANTIOMERS; HANDEDNESS; ABSOLUTE CONFIGURATION; CHIRALITY; TWINNING; EXPERIMENTAL PHASING Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces com­bined real–imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the software Phaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods. text/html Experimental phasing: best practice and pitfalls text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 458 research papers 0907-4449 med@iucr.org 469 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5149 X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Weik, M. Colletier, J.-P. 2010-04-01 doi:10.1107/S0907444910002702 International Union of Crystallography The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. en TEMPERATURE-DEPENDENT MACROMOLECULAR CRYSTALLOGRAPHY X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography. text/html Temperature-dependent macromolecular X-ray crystallography text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 437 research papers 0907-4449 med@iucr.org 446 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5193 Up to 2% of X-ray structures in the Protein Data Bank (PDB) potentially fit into a higher symmetry space group. Redundant protein chains in these structures can be made compatible with exact crystallographic symmetry with minimal atomic movements that are smaller than the expected range of coordinate uncertainty. The incidence of problem cases is somewhat difficult to define precisely, as there is no clear line between underassigned symmetry, in which the subunit differences are unsupported by the data, and pseudosymmetry, in which the subunit differences rest on small but significant intensity differences in the diffraction pattern. To help catch symmetry-assignment problems in the future, it is useful to add a validation step that operates on the refined coordinates just prior to structure deposition. If redundant symmetry-related chains can be removed at this stage, the resulting model (in a higher symmetry space group) can readily serve as an isomorphous replacement starting point for re-refinement using re-indexed and re-integrated raw data. These ideas are implemented in new software tools available at http://cci.lbl.gov/labelit. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Poon, B.K. Grosse-Kunstleve, R.W. Zwart, P.H. Sauter, N.K. 2010-05-01 doi:10.1107/S0907444910001502 International Union of Crystallography An X-ray structural model can be reassigned to a higher symmetry space group using the presented framework if its noncrystallographic symmetry operators are close to being exact crystallographic relationships. About 2% of structures in the Protein Data Bank can be reclassified in this way. en UNDERASSIGNED ROTATIONAL SYMMETRY; LABELIT; VALIDATION Up to 2% of X-ray structures in the Protein Data Bank (PDB) potentially fit into a higher symmetry space group. Redundant protein chains in these structures can be made compatible with exact crystallographic symmetry with minimal atomic movements that are smaller than the expected range of coordinate uncertainty. The incidence of problem cases is somewhat difficult to define precisely, as there is no clear line between underassigned symmetry, in which the subunit differences are unsupported by the data, and pseudosymmetry, in which the subunit differences rest on small but significant intensity differences in the diffraction pattern. To help catch symmetry-assignment problems in the future, it is useful to add a validation step that operates on the refined coordinates just prior to structure deposition. If redundant symmetry-related chains can be removed at this stage, the resulting model (in a higher symmetry space group) can readily serve as an isomorphous replacement starting point for re-refinement using re-indexed and re-integrated raw data. These ideas are implemented in new software tools available at http://cci.lbl.gov/labelit. text/html Detection and correction of underassigned rotational symmetry prior to structure deposition text 66 5 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-05-01 503 research papers 0907-4449 med@iucr.org 513 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5183 A method for rapidly building β-sheets into electron-density maps is presented. β-Strands are identified as tubes of high density adjacent to and nearly parallel to other tubes of density. The alignment and direction of each strand are identified from the pattern of high density corresponding to carbonyl and Cβ atoms along the strand averaged over all repeats present in the strand. The β-strands obtained are then assembled into a single atomic model of the β-sheet regions. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 Å. The β-­sheet regions were nearly completely built in all but two cases, the exceptions being one structure at 2.5 Å resolution in which a third of the residues in β-sheets were built and a structure at 3.8 Å in which under 10% were built. The overall average r.m.s.d. of main-chain atoms in the residues built using this method compared with refined models of the structures was 1.5 Å. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Terwilliger, T.C. 2010-03-01 doi:10.1107/S0907444910000302 International Union of Crystallography A method for rapid model building of β-sheets at moderate resolution is presented. en STRUCTURE SOLUTION; MODEL BUILDING; PROTEIN DATA BANK; [BETA]-STRANDS; PHENIX; EXPERIMENTAL ELECTRON-DENSITY MAPS A method for rapidly building β-sheets into electron-density maps is presented. β-Strands are identified as tubes of high density adjacent to and nearly parallel to other tubes of density. The alignment and direction of each strand are identified from the pattern of high density corresponding to carbonyl and Cβ atoms along the strand averaged over all repeats present in the strand. The β-strands obtained are then assembled into a single atomic model of the β-sheet regions. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 Å. The β-­sheet regions were nearly completely built in all but two cases, the exceptions being one structure at 2.5 Å resolution in which a third of the residues in β-sheets were built and a structure at 3.8 Å in which under 10% were built. The overall average r.m.s.d. of main-chain atoms in the residues built using this method compared with refined models of the structures was 1.5 Å. text/html Rapid model building of β-sheets in electron-density maps text 66 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-03-01 276 research papers 0907-4449 med@iucr.org 284 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5184 A method for the rapid tracing of polypeptide backbones has been developed. The method creates an approximate chain tracing that is useful for visual evaluation of whether a structure has been solved and for use in scoring the quality of electron-density maps. The essence of the method is to (i) sample candidate Cα positions at spacings of approximately 0.6 Å along ridgelines of high electron density, (ii) list all possible nonapeptides that satisfy simple geometric and density criteria using these candidate Cα positions, (iii) score the nonapeptides and choose the highest scoring ones, and (iv) find the longest chains that can be made by connecting nonamers. An indexing and storage scheme that allows a single calculation of most distances and density values is used to speed up the process. The method was applied to 42 density-modified electron-density maps at resolutions from 1.5 to 3.8 Å. A total of 21 428 residues in these maps were traced in 24 CPU min with an overall r.m.s.d. of 1.61 Å for Cα atoms compared with the known refined structures. The method appears to be suitable for rapid evaluation of electron-density map quality. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Terwilliger, T.C. 2010-03-01 doi:10.1107/S0907444910000272 International Union of Crystallography A method for rapid chain tracing of polypeptide backbones at moderate resolution is presented. en STRUCTURE SOLUTION; MODEL BUILDING; PROTEIN DATA BANK; CHAIN TRACING; PHENIX; EXPERIMENTAL ELECTRON-DENSITY MAPS; C[ALPHA] POSITIONS A method for the rapid tracing of polypeptide backbones has been developed. The method creates an approximate chain tracing that is useful for visual evaluation of whether a structure has been solved and for use in scoring the quality of electron-density maps. The essence of the method is to (i) sample candidate Cα positions at spacings of approximately 0.6 Å along ridgelines of high electron density, (ii) list all possible nonapeptides that satisfy simple geometric and density criteria using these candidate Cα positions, (iii) score the nonapeptides and choose the highest scoring ones, and (iv) find the longest chains that can be made by connecting nonamers. An indexing and storage scheme that allows a single calculation of most distances and density values is used to speed up the process. The method was applied to 42 density-modified electron-density maps at resolutions from 1.5 to 3.8 Å. A total of 21 428 residues in these maps were traced in 24 CPU min with an overall r.m.s.d. of 1.61 Å for Cα atoms compared with the known refined structures. The method appears to be suitable for rapid evaluation of electron-density map quality. text/html Rapid chain tracing of polypeptide backbones in electron-density maps text 66 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-03-01 285 research papers 0907-4449 med@iucr.org 294 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5182 A method for the identification of α-helices in electron-density maps at low resolution followed by interpretation at moderate to high resolution is presented. Rapid identification is achieved at low resolution, where α-helices appear as tubes of density. The positioning and direction of the α-helices is obtained at moderate to high resolution, where the positions of side chains can be seen. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 Å. An average of 63% of the α-helical residues in these proteins were built and an average of 76% of the residues built matched helical residues in the refined models of the proteins. The overall average r.m.s.d. between main-chain atoms in the modeled α-helices and the nearest atom with the same name in the refined models of the proteins was 1.3 Å. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Terwilliger, T.C. 2010-03-01 doi:10.1107/S0907444910000314 International Union of Crystallography A method for rapid model building of α-helices at moderate resolution is presented. en STRUCTURE SOLUTION; MODEL BUILDING; PROTEIN DATA BANK; [ALPHA]-HELICES; PHENIX; EXPERIMENTAL ELECTRON-DENSITY MAPS A method for the identification of α-helices in electron-density maps at low resolution followed by interpretation at moderate to high resolution is presented. Rapid identification is achieved at low resolution, where α-helices appear as tubes of density. The positioning and direction of the α-helices is obtained at moderate to high resolution, where the positions of side chains can be seen. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 Å. An average of 63% of the α-helical residues in these proteins were built and an average of 76% of the residues built matched helical residues in the refined models of the proteins. The overall average r.m.s.d. between main-chain atoms in the modeled α-helices and the nearest atom with the same name in the refined models of the proteins was 1.3 Å. text/html Rapid model building of α-helices in electron-density maps text 66 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-03-01 268 research papers 0907-4449 med@iucr.org 275 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5145 To take into account the effects of radiation damage, new algorithms for the optimization of data-collection strategies have been implemented in the software package BEST. The intensity variation related to radiation damage is approximated by log-linear functions of resolution and cumulative X-­ray dose. Based on an accurate prediction of the basic characteristics of data yet to be collected, BEST establishes objective relationships between the accessible data completeness, resolution and signal-to-noise statistics that can be achieved in an experiment and designs an optimal plan for data collection. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Bourenkov, G.P. Popov, A.N. 2010-04-01 doi:10.1107/S0907444909054961 International Union of Crystallography Software implementing a new method for the optimal choice of data-collection parameters, accounting for the effects of radiation damage, is presented. en X-RAY DATA COLLECTION; PROTEIN CRYSTALS; BEST SOFTWARE; RADIATION DAMAGE To take into account the effects of radiation damage, new algorithms for the optimization of data-collection strategies have been implemented in the software package BEST. The intensity variation related to radiation damage is approximated by log-linear functions of resolution and cumulative X-­ray dose. Based on an accurate prediction of the basic characteristics of data yet to be collected, BEST establishes objective relationships between the accessible data completeness, resolution and signal-to-noise statistics that can be achieved in an experiment and designs an optimal plan for data collection. text/html Optimization of data collection taking radiation damage into account text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 409 research papers 0907-4449 med@iucr.org 419 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5140 The space-group symmetry of a crystal structure imposes a point-group symmetry on its diffraction pattern, giving rise to so-called symmetry-equivalent reflections. Instances in macromolecular crystallography are discussed in which the sym­metry in reciprocal space is broken, i.e. where symmetry-related reflections are no longer equivalent. Such a situation occurs when the sample suffers from site-specific radiation damage during the X-ray measurements. Another example of broken symmetry arises from the polarization anisotropy of anomalous scattering. In these cases, the genuine intensity differences between symmetry-related reflections can be exploited to yield phase information in the structure-solution process. In this approach, the usual separation of the data merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking effects are explicitly modelled and refined and become a source of supplementary phase information. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Schiltz, M. Bricogne, G. 2010-04-01 doi:10.1107/S0907444909053578 International Union of Crystallography Site-specific radiation damage and anisotropy of anomalous scattering can induce intensity differences in symmetry-related reflections. If the data are kept unmerged, these symmetry-breaking effects can become a source of phase information. en BROKEN SYMMETRY; PHASING; RADIATION DAMAGE; POLARIZATION ANISOTROPY The space-group symmetry of a crystal structure imposes a point-group symmetry on its diffraction pattern, giving rise to so-called symmetry-equivalent reflections. Instances in macromolecular crystallography are discussed in which the sym­metry in reciprocal space is broken, i.e. where symmetry-related reflections are no longer equivalent. Such a situation occurs when the sample suffers from site-specific radiation damage during the X-ray measurements. Another example of broken symmetry arises from the polarization anisotropy of anomalous scattering. In these cases, the genuine intensity differences between symmetry-related reflections can be exploited to yield phase information in the structure-solution process. In this approach, the usual separation of the data merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking effects are explicitly modelled and refined and become a source of supplementary phase information. text/html `Broken symmetries' in macromolecular crystallography: phasing from unmerged data text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 447 research papers 0907-4449 med@iucr.org 457 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5135 Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the `classical' heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom com­pound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the pro­cess of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Joyce, M.G. Radaev, S. Sun, P.D. 2010-04-01 doi:10.1107/S0907444909053074 International Union of Crystallography In order to overcome the difficulties associated with the `classical' heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. en HEAVY-ATOM DERIVATIVIZATION; HEAVY-ATOM SCREENING; PHASING; STRUCTURE DETERMINATION Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the `classical' heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom com­pound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the pro­cess of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing. text/html A rational approach to heavy-atom derivative screening text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 358 research papers 0907-4449 med@iucr.org 365 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5186 Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Adams, P.D. Afonine, P.V. Bunkóczi, G. Chen, V.B. Davis, I.W. Echols, N. Headd, J.J. Hung, L.-W. Kapral, G.J. Grosse-Kunstleve, R.W. McCoy, A.J. Moriarty, N.W. Oeffner, R. Read, R.J. Richardson, D.C. Richardson, J.S. Terwilliger, T.C. Zwart, P.H. 2010-02-01 doi:10.1107/S0907444909052925 International Union of Crystallography The PHENIX software for macromolecular structure determination is described. en PHENIX; PYTHON; MACROMOLECULAR CRYSTALLOGRAPHY; ALGORITHMS Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms. text/html PHENIX: a comprehensive Python-based system for macromolecular structure solution text 66 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-02-01 213 research papers 0907-4449 med@iucr.org 221 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5137 Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Beck, T. Gruene, T. Sheldrick, G.M. 2010-04-01 doi:10.1107/S0907444909051609 International Union of Crystallography 5-Amino-2,4,6-tribromoisophthalic acid is used as a phasing tool for protein structure determination by MAD phasing. It is the second representative of a novel class of compounds for heavy-atom derivatization that combine heavy atoms with amino and carboxyl groups for binding to proteins. en MULTI-WAVELENGTH ANOMALOUS DISPERSION; EXPERIMENTAL PHASING; HEAVY-ATOM DERIVATIVES Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out. text/html The magic triangle goes MAD: experimental phasing with a bromine derivative text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 374 research papers 0907-4449 med@iucr.org 380 1399-0047 http://scripts.iucr.org/cgi-bin/paper?yt5020 HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (Rwork = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (Rwork = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kim, W.M. Sigalov, A.B. Stern, L.J. 2010-02-01 doi:10.1107/S090744490904880X International Union of Crystallography P212121 crystals of SIV Nef core domain bound to a peptide fragment of the T-cell receptor ζ subunit exhibited noncrystallographic symmetry and nearly perfect pseudo-merohedral twinning simulating tetragonal symmetry. For a different peptide fragment, nontwinned tetragonal crystals were observed but diffracted to lower resolution. The structure was determined after assignment of the top molecular-replacement solutions to various twin or NCS domains followed by refinement under the appropriate twin law. en PSEUDO-MEROHEDRAL TWINNING; NONCRYSTALLOGRAPHIC SYMMETRY; PSEUDOSYMMETRY; HUMAN IMMUNODEFICIENCY VIRUS; NEF; T-CELL RECEPTOR HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (Rwork = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (Rwork = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning. text/html Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments text 66 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-02-01 163 research papers 0907-4449 med@iucr.org 175 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5141 In isolation, both weak isomorphous/anomalous difference signals from heavy-atom derivatization and phases from partial molecular-replacement solutions for a subset of the asymmetric unit often fall short of producing interpretable electron-density maps. Phases generated from very partial molecular-replacement models (if generated carefully) can be used to reliably locate heavy-atom sites, even if the signal is not sufficiently strong to allow robust finding of the sites using Patterson interpretation or direct methods. Additional advantages are that using molecular-replacement phases to define the heavy-atom substructure avoids the need for subsequent hand determination and/or origin-choice reconciliation and that the partial model can be used to aid the mask determination during solvent flattening. Two case studies are presented in which it was only by combining experimental and molecular-replacement phasing approaches that the crystal structures could be determined. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Roversi, P. Johnson, S. Lea, S.M. 2010-04-01 doi:10.1107/S0907444909048112 International Union of Crystallography Combining experimental phases and those from refinement of very incomplete models significantly improves electron-density maps. en MOLECULAR REPLACEMENT; EXPERIMENTAL PHASING; PHASE COMBINATION In isolation, both weak isomorphous/anomalous difference signals from heavy-atom derivatization and phases from partial molecular-replacement solutions for a subset of the asymmetric unit often fall short of producing interpretable electron-density maps. Phases generated from very partial molecular-replacement models (if generated carefully) can be used to reliably locate heavy-atom sites, even if the signal is not sufficiently strong to allow robust finding of the sites using Patterson interpretation or direct methods. Additional advantages are that using molecular-replacement phases to define the heavy-atom substructure avoids the need for subsequent hand determination and/or origin-choice reconciliation and that the partial model can be used to aid the mask determination during solvent flattening. Two case studies are presented in which it was only by combining experimental and molecular-replacement phasing approaches that the crystal structures could be determined. text/html With phases: how two wrongs can sometimes make a right text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 420 research papers 0907-4449 med@iucr.org 425 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5178 Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-reduction package XDS are explained, including the new features of automatic determination of spot size and reflecting range, recognition and assignment of crystal sym­metry and a highly efficient algorithm for the determination of correction/scaling factors. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kabsch, W. 2010-02-01 doi:10.1107/S0907444909047374 International Union of Crystallography The working principles of important steps in processing rotation data are described as employed by the program XDS. en XDS; INTEGRATION; SCALING; SPACE-GROUP ASSIGNMENT; POST-REFINEMENT Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-reduction package XDS are explained, including the new features of automatic determination of spot size and reflecting range, recognition and assignment of crystal sym­metry and a highly efficient algorithm for the determination of correction/scaling factors. text/html Integration, scaling, space-group assignment and post-refinement text 66 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-02-01 133 research papers 0907-4449 med@iucr.org 144 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5179 The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic determination of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the number of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kabsch, W. 2010-02-01 doi:10.1107/S0907444909047337 International Union of Crystallography The paper describes the software package XDS for processing of single crystal diffraction data recorded by the rotation method. en XDS The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic determination of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the number of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement. text/html XDS text 66 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-02-01 125 research papers 0907-4449 med@iucr.org 132 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5180 MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallo­graphy easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Chen, V.B. Arendall, W.B. Headd, J.J. Keedy, D.A. Immormino, R.M. Kapral, G.J. Murray, L.W. Richardson, J.S. Richardson, D.C. 2010-01-01 doi:10.1107/S0907444909042073 International Union of Crystallography MolProbity structure validation will diagnose most local errors in macromolecular crystal structures and help to guide their correction. en ALL-ATOM CONTACTS; CLASHSCORE; AUTOMATED CORRECTION; KING; RIBOSE PUCKER; RAMACHANDRAN PLOTS; SIDE-CHAIN ROTAMERS; MODEL QUALITY; SYSTEMATIC ERRORS; DATABASE IMPROVEMENT MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallo­graphy easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database. text/html MolProbity: all-atom structure validation for macromolecular crystallography text 66 1 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-01-01 12 research papers 0907-4449 med@iucr.org 21 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5139 In macromolecular crystallography, the acquisition of a complete set of diffraction intensities typically involves a high cumulative dose of X-ray radiation. In the process of data acquisition, the irradiated crystal lattice undergoes a broad range of chemical and physical changes. These result in the gradual decay of diffraction intensities, accompanied by changes in the macroscopic organization of crystal lattice order and by localized changes in electron density that, owing to complex radiation chemistry, are specific for a particular macromolecule. The decay of diffraction intensities is a well defined physical process that is fully correctable during scaling and merging analysis and therefore, while limiting the amount of diffraction, it has no other impact on phasing procedures. Specific chemical changes, which are variable even between different crystal forms of the same macromolecule, are more difficult to predict, describe and correct in data. Appearing during the process of data collection, they result in gradual changes in structure factors and therefore have profound consequences in phasing procedures. Examples of various combinations of radiation-induced changes are presented and various considerations pertinent to the determination of the best strategies for handling diffraction data analysis in representative situations are discussed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Borek, D. Cymborowski, M. Machius, M. Minor, W. Otwinowski, Z. 2010-04-01 doi:10.1107/S0907444909040177 International Union of Crystallography Radiation-induced decay of crystal diffraction and additional specific chemical changes of macromolecules forming the crystal lattice are currently two of the main limiting factors in the acquisition of macromolecular diffraction data and macromolecular structure determination. Data-processing and phasing protocols are discussed in the context of radiation-induced changes. en RADIATION-INDUCED SPECIFIC CHANGES; RELATIVE B FACTOR; SCALING B FACTOR; EXPERIMENTAL PHASING; SYNCHROTRON RADIATION In macromolecular crystallography, the acquisition of a complete set of diffraction intensities typically involves a high cumulative dose of X-ray radiation. In the process of data acquisition, the irradiated crystal lattice undergoes a broad range of chemical and physical changes. These result in the gradual decay of diffraction intensities, accompanied by changes in the macroscopic organization of crystal lattice order and by localized changes in electron density that, owing to complex radiation chemistry, are specific for a particular macromolecule. The decay of diffraction intensities is a well defined physical process that is fully correctable during scaling and merging analysis and therefore, while limiting the amount of diffraction, it has no other impact on phasing procedures. Specific chemical changes, which are variable even between different crystal forms of the same macromolecule, are more difficult to predict, describe and correct in data. Appearing during the process of data collection, they result in gradual changes in structure factors and therefore have profound consequences in phasing procedures. Examples of various combinations of radiation-induced changes are presented and various considerations pertinent to the determination of the best strategies for handling diffraction data analysis in representative situations are discussed. text/html Diffraction data analysis in the presence of radiation damage text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 426 research papers 0907-4449 med@iucr.org 436 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5138 Cryogenic cooling of macromolecular crystals is commonly used for X-ray data collection both to reduce crystal damage from radiation and to gather functional information by cryogenically trapping intermediates. However, the cooling process can damage the crystals. Limiting cooling-induced crystal damage often requires cryoprotection strategies, which can involve substantial screening of solution conditions and cooling protocols. Here, recent developments directed towards rational methods for cryoprotection are described. Crystal damage is described in the context of the temperature response of the crystal as a thermodynamic system. As such, the internal and external parts of the crystal typically have different cryoprotection requirements. A key physical parameter, the thermal contraction, of 26 different cryoprotective solutions was measured between 294 and 72 K. The range of contractions was 2–13%, with the more polar cryosolutions contracting less. The potential uses of these results in the development of cryocooling conditions, as well as recent developments in determining minimum cryosolution soaking times, are discussed. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Alcorn, T. Juers, D.H. 2010-04-01 doi:10.1107/S090744490903995X International Union of Crystallography Measurements of the average thermal contractions (294→72 K) of 26 different cryosolutions are presented and discussed in conjunction with other recent advances in the rational design of protocols for cryogenic cooling in macromolecular crystallography. en CRYOPROTECTION; CRYOSOLUTIONS; THERMAL CONTRACTION; CRYSTAL DAMAGE; DOMAIN STRUCTURE Cryogenic cooling of macromolecular crystals is commonly used for X-ray data collection both to reduce crystal damage from radiation and to gather functional information by cryogenically trapping intermediates. However, the cooling process can damage the crystals. Limiting cooling-induced crystal damage often requires cryoprotection strategies, which can involve substantial screening of solution conditions and cooling protocols. Here, recent developments directed towards rational methods for cryoprotection are described. Crystal damage is described in the context of the temperature response of the crystal as a thermodynamic system. As such, the internal and external parts of the crystal typically have different cryoprotection requirements. A key physical parameter, the thermal contraction, of 26 different cryoprotective solutions was measured between 294 and 72 K. The range of contractions was 2–13%, with the more polar cryosolutions contracting less. The potential uses of these results in the development of cryocooling conditions, as well as recent developments in determining minimum cryosolution soaking times, are discussed. text/html Progress in rational methods of cryoprotection in macromolecular crystallography text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 366 research papers 0907-4449 med@iucr.org 373 1399-0047 http://scripts.iucr.org/cgi-bin/paper?lv5009 Predictions of the possible model parameterization and of the values of model characteristics such as R factors are important for macromolecular refinement and validation protocols. One of the key parameters defining these and other values is the resolution of the experimentally measured diffraction data. The higher the resolution, the larger the number of diffraction data Nref, the larger its ratio to the number Nat of non-H atoms, the more parameters per atom can be used for modelling and the more precise and detailed a model can be obtained. The ratio Nref/Nat was calculated for models deposited in the Protein Data Bank as a function of the resolution at which the structures were reported. The most frequent values for this distribution depend essentially linearly on resolution when the latter is expressed on a uniform logarithmic scale. This defines simple analytic formulae for the typical Matthews coefficient and for the typically allowed number of parameters per atom for crystals diffracting to a given resolution. This simple dependence makes it possible in many cases to estimate the expected resolution of the experimental data for a crystal with a given Matthews coefficient. When expressed using the same logarithmic scale, the most frequent values for R and Rfree factors and for their difference are also essentially linear across a large resolution range. The minimal R-factor values are practically constant at resolutions better than 3 Å, below which they begin to grow sharply. This simple dependence on the resolution allows the prediction of expected R-factor values for unknown structures and may be used to guide model refinement and validation. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Urzhumtsev, A. Afonine, P.V. Adams, P.D. 2009-12-01 doi:10.1107/S0907444909039638 International Union of Crystallography Conventional and free R factors and their difference, as well as the ratio of the number of measured reflections to the number of atoms in the crystal, were studied as functions of the resolution at which the structures were reported. When the resolution was taken uniformly on a logarithmic scale, the most frequent values of these functions were quasi-linear over a large resolution range. en RESOLUTION; LOGARITHMIC SCALE; R FACTOR; DATA-TO-PARAMETER RATIO Predictions of the possible model parameterization and of the values of model characteristics such as R factors are important for macromolecular refinement and validation protocols. One of the key parameters defining these and other values is the resolution of the experimentally measured diffraction data. The higher the resolution, the larger the number of diffraction data Nref, the larger its ratio to the number Nat of non-H atoms, the more parameters per atom can be used for modelling and the more precise and detailed a model can be obtained. The ratio Nref/Nat was calculated for models deposited in the Protein Data Bank as a function of the resolution at which the structures were reported. The most frequent values for this distribution depend essentially linearly on resolution when the latter is expressed on a uniform logarithmic scale. This defines simple analytic formulae for the typical Matthews coefficient and for the typically allowed number of parameters per atom for crystals diffracting to a given resolution. This simple dependence makes it possible in many cases to estimate the expected resolution of the experimental data for a crystal with a given Matthews coefficient. When expressed using the same logarithmic scale, the most frequent values for R and Rfree factors and for their difference are also essentially linear across a large resolution range. The minimal R-factor values are practically constant at resolutions better than 3 Å, below which they begin to grow sharply. This simple dependence on the resolution allows the prediction of expected R-factor values for unknown structures and may be used to guide model refinement and validation. text/html On the use of logarithmic scales for analysis of diffraction data text 65 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-12-01 1283 research papers 0907-4449 med@iucr.org 1291 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5136 Classical density-modification techniques (as opposed to statistical approaches) offer a computationally cheap method for improving phase estimates in order to provide a good electron-density map for model building. The rise of statistical methods has lead to a shift in focus away from the classical approaches; as a result, some recent developments have not made their way into classical density-modification software. This paper describes the application of some recent tech­niques, including most importantly the use of prior phase information in the likelihood estimation of phase errors within a classical density-modification framework. The resulting software gives significantly better results than comparable classical methods, while remaining nearly two orders of magnitude faster than statistical methods. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Cowtan, K. 2010-04-01 doi:10.1107/S090744490903947X International Union of Crystallography Several new methods are evaluated for use in the improvement of experimental phases in the framework of a classical density-modification calculation. These methods have been implemented in a new computer program, Parrot. en DENSITY MODIFICATION; PHASE IMPROVEMENT; PARROT Classical density-modification techniques (as opposed to statistical approaches) offer a computationally cheap method for improving phase estimates in order to provide a good electron-density map for model building. The rise of statistical methods has lead to a shift in focus away from the classical approaches; as a result, some recent developments have not made their way into classical density-modification software. This paper describes the application of some recent tech­niques, including most importantly the use of prior phase information in the likelihood estimation of phase errors within a classical density-modification framework. The resulting software gives significantly better results than comparable classical methods, while remaining nearly two orders of magnitude faster than statistical methods. text/html Recent developments in classical density modification text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 470 research papers 0907-4449 med@iucr.org 478 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5134 Diffraction data collection is the last experimental stage in structural crystallography. It has several technical and theor­etical aspects and a compromise usually has to be found between various parameters in order to achieve optimal data quality. The influence and importance of various experimental parameters and their consequences are discussed in the context of different data applications, such as molecular replacement, anomalous phasing, high-resolution refinement or searching for ligands. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Dauter, Z. 2010-04-01 doi:10.1107/S0907444909038578 International Union of Crystallography Diffraction data collection parameters leading to optimal data quality are discussed in the context of different applications of these data. en DIFFRACTION DATA COLLECTION; DATA-COLLECTION STRATEGIES; DIFFRACTION EXPERIMENTS Diffraction data collection is the last experimental stage in structural crystallography. It has several technical and theor­etical aspects and a compromise usually has to be found between various parameters in order to achieve optimal data quality. The influence and importance of various experimental parameters and their consequences are discussed in the context of different data applications, such as molecular replacement, anomalous phasing, high-resolution refinement or searching for ligands. text/html Carrying out an optimal experiment text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 389 research papers 0907-4449 med@iucr.org 392 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5143 The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue α-­helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a `no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Sheldrick, G.M. 2010-04-01 doi:10.1107/S0907444909038360 International Union of Crystallography Experimental phasing with SHELXC/D/E has been enhanced by the incorporation of main-chain tracing into the iterative density modification; this also provides a simple and effective way of exploiting noncrystallographic symmetry. en EXPERIMENTAL PHASING OF MACROMOLECULES; DENSITY MODIFICATION; MAIN-CHAIN TRACING; NONCRYSTALLOGRAPHIC SYMMETRY; SHELX The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue α-­helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a `no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap. text/html Experimental phasing with SHELXC/D/E: combining chain tracing with density modification text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 479 research papers 0907-4449 med@iucr.org 485 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5133 Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in pro­karyal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Walden, H. 2010-04-01 doi:10.1107/S0907444909038207 International Union of Crystallography An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. en SELENIUM INCORPORATION; SELENIUM LABELLING Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in pro­karyal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included. text/html Selenium incorporation using recombinant techniques text 66 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-04-01 352 research papers 0907-4449 med@iucr.org 357 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5061 Charge-coupled devices (CCD) are nowadays commonly utilized in transmission electron microscopy (TEM) for applications in life sciences. Direct access to digitized images has revolutionized the use of electron microscopy, sparking developments such as automated collection of tomographic data, focal series, random conical tilt pairs and ultralarge single-particle data sets. Nevertheless, for ultrahigh-resolution work photographic plates are often still preferred. In the ideal case, the quality of the recorded image of a vitrified biological sample would solely be determined by the counting statistics of the limited electron dose the sample can withstand before beam-induced alterations dominate. Unfortunately, the image is degraded by the non-ideal point-spread function of the detector, as a result of a scintillator coupled by fibre optics to a CCD, and the addition of several inherent noise components. Different detector manufacturers provide different types of figures of merit when advertising the quality of their detector. It is hard for most laboratories to verify whether all of the anticipated specifications are met. In this report, a set of algorithms is presented to characterize on-axis slow-scan large-area CCD-based TEM detectors. These tools have been added to a publicly available image-processing toolbox for MATLAB. Three in-house CCD cameras were carefully characterized, yielding, among others, statistics for hot and bad pixels, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. These statistics will aid data-collection strategy programs and provide prior information for quantitative imaging. The relative performance of the characterized detectors is discussed and a comparison is made with similar detectors that are used in the field of X-ray crystallography. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Vulovic, M. Rieger, B. van Vliet, L.J. Koster, A.J. Ravelli, R.B.G. 2010-01-01 doi:10.1107/S0907444909031205 International Union of Crystallography A toolkit for the characterization of CCD cameras has been developed. Its use has been demonstrated by the characterization of three in-house transmission electron microscopy detectors, yielding statistics such as the number of outliers, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. en CCD CAMERAS; TRANSMISSION ELECTRON MICROSCOPY; PIXEL-ARRAY DETECTORS Charge-coupled devices (CCD) are nowadays commonly utilized in transmission electron microscopy (TEM) for applications in life sciences. Direct access to digitized images has revolutionized the use of electron microscopy, sparking developments such as automated collection of tomographic data, focal series, random conical tilt pairs and ultralarge single-particle data sets. Nevertheless, for ultrahigh-resolution work photographic plates are often still preferred. In the ideal case, the quality of the recorded image of a vitrified biological sample would solely be determined by the counting statistics of the limited electron dose the sample can withstand before beam-induced alterations dominate. Unfortunately, the image is degraded by the non-ideal point-spread function of the detector, as a result of a scintillator coupled by fibre optics to a CCD, and the addition of several inherent noise components. Different detector manufacturers provide different types of figures of merit when advertising the quality of their detector. It is hard for most laboratories to verify whether all of the anticipated specifications are met. In this report, a set of algorithms is presented to characterize on-axis slow-scan large-area CCD-based TEM detectors. These tools have been added to a publicly available image-processing toolbox for MATLAB. Three in-house CCD cameras were carefully characterized, yielding, among others, statistics for hot and bad pixels, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. These statistics will aid data-collection strategy programs and provide prior information for quantitative imaging. The relative performance of the characterized detectors is discussed and a comparison is made with similar detectors that are used in the field of X-ray crystallography. text/html A toolkit for the characterization of CCD cameras for transmission electron microscopy text 66 1 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2010-01-01 97 research papers 0907-4449 med@iucr.org 109 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ea5105 The electronic Ligand Builder and Optimization Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallographic software. It is designed to be a flexible procedure that uses simple and fast quantum-chemical techniques to provide chemically accurate information for novel and known ligands alike. A variety of input formats and options allow the attainment of a number of diverse goals including geometry optimization and generation of restraints. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Moriarty, N.W. Grosse-Kunstleve, R.W. Adams, P.D. 2009-10-01 doi:10.1107/S0907444909029436 International Union of Crystallography A new software system for automated ligand coordinate and restraint generation is presented. en LIGANDS; COORDINATES; RESTRAINTS; PYTHON; OBJECT-ORIENTED PROGRAMMING The electronic Ligand Builder and Optimization Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallographic software. It is designed to be a flexible procedure that uses simple and fast quantum-chemical techniques to provide chemically accurate information for novel and known ligands alike. A variety of input formats and options allow the attainment of a number of diverse goals including geometry optimization and generation of restraints. text/html electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation text 65 10 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-10-01 1074 research papers 0907-4449 med@iucr.org 1080 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5288 When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kors, C.A. Wallace, E. Davies, D.R. Li, L. Laible, P.D. Nollert, P. 2009-10-01 doi:10.1107/S0907444909029163 International Union of Crystallography The effects of commonly encountered impurities on various membrane-protein crystallization regimes are investigated and it is found that the lipidic cubic phase crystallization methodology is the most robust, tolerating protein contamination levels of up to 50%, with little effect on crystal quality. If generally applicable, this tolerance may be exploited (i) in initial crystallization trials to determine the `crystallizability' of a given membrane-protein and (ii) to subject partially pure membrane-protein samples to crystallization trials. en MEMBRANE PROTEINS; IMPURITIES; CRYSTALLIZATION When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein. text/html Effects of impurities on membrane-protein crystallization in different systems text 65 10 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-10-01 1062 research papers 0907-4449 med@iucr.org 1073 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ea5102 A likelihood function based on the multivariate probability distribution of all observed structure-factor amplitudes from a single isomorphous replacement with anomalous scattering experiment has been derived and implemented for use in substructure refinement and phasing as well as macromolecular model refinement. Efficient calculation of a multidimensional integration required for function evaluation has been achieved by approximations based on the function's properties. The use of the function in both phasing and protein model building with iterative refinement was essential for successful automated model building in the test cases presented. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Skubák, P. Murshudov, G. Pannu, N.S. 2009-10-01 doi:10.1107/S0907444909028078 International Union of Crystallography The application of a multivariate likelihood function to a single isomorphous replacement with anomalous scattering experiment improves phasing and automated model building with iterative refinement in the test cases shown. en MULTIVARIATE NORMAL PROBABILITY DISTRIBUTION; SINGLE ISOMORPHOUS REPLACEMENT WITH ANOMALOUS SCATTERING; EXPERIMENTAL PHASING; DIRECT INCORPORATION OF PRIOR PHASE INFORMATION A likelihood function based on the multivariate probability distribution of all observed structure-factor amplitudes from a single isomorphous replacement with anomalous scattering experiment has been derived and implemented for use in substructure refinement and phasing as well as macromolecular model refinement. Efficient calculation of a multidimensional integration required for function evaluation has been achieved by approximations based on the function's properties. The use of the function in both phasing and protein model building with iterative refinement was essential for successful automated model building in the test cases presented. text/html A multivariate likelihood SIRAS function for phasing and model refinement text 65 10 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-10-01 1051 research papers 0907-4449 med@iucr.org 1061 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5169 A response is published to the comment by Okuyama et al. [(2009) Acta Cryst. D65, 1007–1008] on Microfibrillar structure of type I collagen in situ. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Orgel, J.P.R.O. 2009-09-01 doi:10.1107/S0907444909028741 International Union of Crystallography A response to the comment on Microfibrillar structure of type I collagen in situ [Okuyama et al. (2009), Acta Cryst. D65, 1007–1008]. en LETTERS TO THE EDITOR; TYPE I COLLAGEN A response is published to the comment by Okuyama et al. [(2009) Acta Cryst. D65, 1007–1008] on Microfibrillar structure of type I collagen in situ. text/html On the packing structure of collagen: response to Okuyama et al.'s comment on Microfibrillar structure of type I collagen in situ text 65 9 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-09-01 1009 letters to the editor 0907-4449 med@iucr.org 1010 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5164 Recent advances in computational chemistry have produced force fields based on a polarizable atomic multipole description of biomolecular electrostatics. In this work, the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field is applied to restrained refinement of molecular models against X-ray diffraction data from peptide crystals. A new formalism is also developed to compute anisotropic and aspherical structure factors using fast Fourier transformation (FFT) of Cartesian Gaussian multipoles. Relative to direct summation, the FFT approach can give a speedup of more than an order of magnitude for aspherical refinement of ultrahigh-resolution data sets. Use of a sublattice formalism makes the method highly parallelizable. Application of the Cartesian Gaussian multipole scattering model to a series of four peptide crystals using multipole coefficients from the AMOEBA force field demonstrates that AMOEBA systematically underestimates electron density at bond centers. For the trigonal and tetrahedral bonding geometries common in organic chemistry, an atomic multipole expansion through hexadecapole order is required to explain bond electron density. Alternatively, the addition of inter­atomic scattering (IAS) sites to the AMOEBA-based density captured bonding effects with fewer parameters. For a series of four peptide crystals, the AMOEBA–IAS model lowered Rfree by 20–40% relative to the original spherically symmetric scattering model. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Schnieders, M.J. Fenn, T.D. Pande, V.S. Brunger, A.T. 2009-09-01 doi:10.1107/S0907444909022707 International Union of Crystallography A method to accelerate the computation of structure factors from an electron density described by anisotropic and aspherical atomic form factors via fast Fourier transformation is described for the first time. en SCATTERING FACTORS; ASPHERICAL; ANISOTROPIC; FORCE FIELDS; MULTIPOLE; POLARIZATION; AMOEBA; BOND DENSITY; DIRECT SUMMATION; FFT; SGFFT; EWALD; PME Recent advances in computational chemistry have produced force fields based on a polarizable atomic multipole description of biomolecular electrostatics. In this work, the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field is applied to restrained refinement of molecular models against X-ray diffraction data from peptide crystals. A new formalism is also developed to compute anisotropic and aspherical structure factors using fast Fourier transformation (FFT) of Cartesian Gaussian multipoles. Relative to direct summation, the FFT approach can give a speedup of more than an order of magnitude for aspherical refinement of ultrahigh-resolution data sets. Use of a sublattice formalism makes the method highly parallelizable. Application of the Cartesian Gaussian multipole scattering model to a series of four peptide crystals using multipole coefficients from the AMOEBA force field demonstrates that AMOEBA systematically underestimates electron density at bond centers. For the trigonal and tetrahedral bonding geometries common in organic chemistry, an atomic multipole expansion through hexadecapole order is required to explain bond electron density. Alternatively, the addition of inter­atomic scattering (IAS) sites to the AMOEBA-based density captured bonding effects with fewer parameters. For a series of four peptide crystals, the AMOEBA–IAS model lowered Rfree by 20–40% relative to the original spherically symmetric scattering model. text/html Polarizable atomic multipole X-ray refinement: application to peptide crystals text 65 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-09-01 952 research papers 0907-4449 med@iucr.org 965 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5129 Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an Rcryst of 19.0% and an Rfree of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described pre­viously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kirkensgaard, K.G. Hägglund, P. Finnie, C. Svensson, B. Henriksen, A. 2009-09-01 doi:10.1107/S0907444909021817 International Union of Crystallography The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. en NADPH-DEPENDENT THIOREDOXIN REDUCTASES; DISULFIDE OXIDOREDUCTASES; BARLEY; GERMINATION; SEED DEVELOPMENT; NADPH; REDOX REGULATION Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an Rcryst of 19.0% and an Rfree of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described pre­viously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation. text/html Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism text 65 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-09-01 932 research papers 0907-4449 med@iucr.org 941 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5160 Use of reliable density maps is crucial for rapid and successful crystal structure determination. Here, the averaged kick (AK) map approach is investigated, its application is generalized and it is compared with other map-calculation methods. AK maps are the sum of a series of kick maps, where each kick map is calculated from atomic coordinates modified by random shifts. As such, they are a numerical analogue of maximum-likelihood maps. AK maps can be unweighted or maximum-likelihood (σA) weighted. Analysis shows that they are comparable and correspond better to the final model than σA and simulated-annealing maps. The AK maps were challenged by a difficult structure-validation case, in which they were able to clarify the problematic region in the density without the need for model rebuilding. The conclusion is that AK maps can be useful throughout the entire progress of crystal structure determination, offering the possibility of improved map interpretation. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Pražnikar, J. Afonine, P.V. Gunčar, G. Adams, P.D. Turk, D. 2009-09-01 doi:10.1107/S0907444909021933 International Union of Crystallography Averaged kick maps are the sum of a series of individual kick maps, where each map is calculated from atomic coordinates modified by random shifts. These maps offer the possibility of an improved and less model-biased map interpretation. en KICK MAPS; OMIT MAPS; DENSITY-MAP CALCULATION; MODEL BIAS; MAXIMUM LIKELIHOOD Use of reliable density maps is crucial for rapid and successful crystal structure determination. Here, the averaged kick (AK) map approach is investigated, its application is generalized and it is compared with other map-calculation methods. AK maps are the sum of a series of kick maps, where each kick map is calculated from atomic coordinates modified by random shifts. As such, they are a numerical analogue of maximum-likelihood maps. AK maps can be unweighted or maximum-likelihood (σA) weighted. Analysis shows that they are comparable and correspond better to the final model than σA and simulated-annealing maps. The AK maps were challenged by a difficult structure-validation case, in which they were able to clarify the problematic region in the density without the need for model rebuilding. The conclusion is that AK maps can be useful throughout the entire progress of crystal structure determination, offering the possibility of improved map interpretation. text/html Averaged kick maps: less noise, more signal…and probably less bias text 65 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-09-01 921 research papers 0907-4449 med@iucr.org 931 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5161 A comment is published on the article Microfibrillar structure of type I collagen in situ by Orgel et al. [(2006), Proc. Natl Acad. Sci. USA, 103, 9001–9005]. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Okuyama, K. Bächinger, H.P. Mizuno, K. Boudko, S. Engel, J. Berisio, R. Vitagliano, L. 2009-09-01 doi:10.1107/S0907444909023051 International Union of Crystallography Comment on Microfibrillar structure of type I collagen in situ by Orgel et al. (2006), Proc. Natl Acad. Sci. USA, 103, 9001–9005. en LETTERS TO THE EDITOR; TYPE I COLLAGEN A comment is published on the article Microfibrillar structure of type I collagen in situ by Orgel et al. [(2006), Proc. Natl Acad. Sci. USA, 103, 9001–9005]. text/html Comment on Microfibrillar structure of type I collagen in situ by Orgel et al. (2006), Proc. Natl Acad. Sci. USA, 103, 9001–9005 text 65 9 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-09-01 1007 letters to the editor 0907-4449 med@iucr.org 1008 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5287 Microfluidics is a promising technology for the rapid iden­tification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Emamzadah, S. Petty, T.J. De Almeida, V. Nishimura, T. Joly, J. Ferrer, J.-L. Halazonetis, T.D. 2009-09-01 doi:10.1107/S0907444909021489 International Union of Crystallography A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. en CYCLIC OLEFIN HOMOPOLYMERS; MICROFLUIDICS; CRYSTALLIZATION; IN SITU X-RAY DIFFRACTION Microfluidics is a promising technology for the rapid iden­tification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts. text/html Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction text 65 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-09-01 913 research papers 0907-4449 med@iucr.org 920 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5133 The controller protein of the Esp1396I restriction–modification (R–M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endo­nuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43–46 in the loop region flanking the DNA-recognition helix are displaced by up to 10 Å. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4 Å displacement of the recognition helices when the dimer is bound to its DNA target. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Ball, N. Streeter, S.D. Kneale, G.G. McGeehan, J.E. 2009-09-01 doi:10.1107/S0907444909020514 International Union of Crystallography The crystal structure of the free restriction–modification controller protein C.Esp1396I is reported. en TRANSCRIPTIONAL REGULATION; RESTRICTION-MODIFICATION SYSTEMS; DNA-BINDING PROTEINS; HELIX-TURN-HELIX MOTIFS The controller protein of the Esp1396I restriction–modification (R–M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endo­nuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43–46 in the loop region flanking the DNA-recognition helix are displaced by up to 10 Å. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4 Å displacement of the recognition helices when the dimer is bound to its DNA target. text/html Structure of the restriction–modification controller protein C.Esp1396I text 65 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-09-01 900 research papers 0907-4449 med@iucr.org 905 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5060 A novel approach to obtaining structural information from macromolecular X-ray data extending to resolutions as low as 20 Å is presented. Following a simple map-segmentation procedure, the approximate shapes of the domains forming the structure are identified. A pattern-recognition comparative analysis of these shapes and those derived from the structures of domains from the PDB results in candidate structural models that can be used for a fit into the density map. It is shown that the placed candidate models can be employed for subsequent phase extension to higher resolution. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Heuser, P. Langer, G.G. Lamzin, V.S. 2009-07-01 doi:10.1107/S090744490901991X International Union of Crystallography The interpretation of a 20 Å resolution electron-density map using segmentation and pattern-recognition-based identification of domain shapes is described. en CORE OBJECTS; LOW-RESOLUTION ELECTRON-DENSITY MAPS; MAP SEGMENTATION; PATTERN RECOGNITION A novel approach to obtaining structural information from macromolecular X-ray data extending to resolutions as low as 20 Å is presented. Following a simple map-segmentation procedure, the approximate shapes of the domains forming the structure are identified. A pattern-recognition comparative analysis of these shapes and those derived from the structures of domains from the PDB results in candidate structural models that can be used for a fit into the density map. It is shown that the placed candidate models can be employed for subsequent phase extension to higher resolution. text/html Interpretation of very low resolution X-ray electron-density maps using core objects text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 690 research papers 0907-4449 med@iucr.org 696 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5162 Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Manolaridis, I. Wojdyla, J.A. Panjikar, S. Snijder, E.J. Gorbalenya, A.E. Berglind, H. Nordlund, P. Coutard, B. Tucker, P.A. 2009-08-01 doi:10.1107/S0907444909018253 International Union of Crystallography The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. en CORONAVIRUSES; NSP4; NONSTRUCTURAL PROTEINS Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions. text/html Structure of the C-terminal domain of nsp4 from feline coronavirus text 65 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-08-01 839 research papers 0907-4449 med@iucr.org 846 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5021 Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor family, which is defined as transcriptional factors that are activated by the binding of ligands to their ligand-binding domains (LBDs). Although the three PPAR subtypes display different tissue distribution patterns and distinct pharmacological profiles, they all are essentially related to fatty-acid and glucose metabolism. Since the PPARs share similar three-dimensional structures within the LBDs, synthetic ligands which simultaneously activate two or all of the PPARs could be potent candidates in terms of drugs for the treatment of abnormal metabolic homeostasis. The structures of several PPAR LBDs were determined in complex with synthetic ligands, derivatives of 3-(4-alkoxy­phenyl)propanoic acid, which exhibit unique agonistic activities. The PPARα and PPARγ LBDs were complexed with the same pan agonist, TIPP-703, which activates all three PPARs and their crystal structures were determined. The two LBD–ligand complex structures revealed how the pan agonist is adapted to the similar, but significantly different, ligand-binding pockets of the PPARs. The structures of the PPARδ LBD in complex with an α/δ-selective ligand, TIPP-401, and with a related δ-­specific ligand, TIPP-204, were also determined. The comparison between the two PPARδ complexes revealed how each ligand exhibits either a `dual selective' or `single specific' binding mode. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Oyama, T. Toyota, K. Waku, T. Hirakawa, Y. Nagasawa, N. Kasuga, J. Hashimoto, Y. Miyachi, H. Morikawa, K. 2009-08-01 doi:10.1107/S0907444909015935 International Union of Crystallography The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level. en PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS; LIGAND-BINDING DOMAINS; AGONISTS Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor family, which is defined as transcriptional factors that are activated by the binding of ligands to their ligand-binding domains (LBDs). Although the three PPAR subtypes display different tissue distribution patterns and distinct pharmacological profiles, they all are essentially related to fatty-acid and glucose metabolism. Since the PPARs share similar three-dimensional structures within the LBDs, synthetic ligands which simultaneously activate two or all of the PPARs could be potent candidates in terms of drugs for the treatment of abnormal metabolic homeostasis. The structures of several PPAR LBDs were determined in complex with synthetic ligands, derivatives of 3-(4-alkoxy­phenyl)propanoic acid, which exhibit unique agonistic activities. The PPARα and PPARγ LBDs were complexed with the same pan agonist, TIPP-703, which activates all three PPARs and their crystal structures were determined. The two LBD–ligand complex structures revealed how the pan agonist is adapted to the similar, but significantly different, ligand-binding pockets of the PPARs. The structures of the PPARδ LBD in complex with an α/δ-selective ligand, TIPP-401, and with a related δ-­specific ligand, TIPP-204, were also determined. The comparison between the two PPARδ complexes revealed how each ligand exhibits either a `dual selective' or `single specific' binding mode. text/html Adaptability and selectivity of human peroxisome proliferator-activated receptor (PPAR) pan agonists revealed from crystal structures text 65 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-08-01 786 research papers 0907-4449 med@iucr.org 795 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5019 Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural deter­mination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Suga, M. Maeda, S. Nakagawa, S. Yamashita, E. Tsukihara, T. 2009-08-01 doi:10.1107/S0907444909014711 International Union of Crystallography The structural determination procedures of a gap junction channel at 3.5 Å resolution are described, including the preparation of crystals, intensity data collection, data processing, phasing and structural refinement. en GAP JUNCTION CHANNEL; MULTI-CRYSTAL AVERAGING; MEMBRANE PROTEINS Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural deter­mination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. text/html A description of the structural determination procedures of a gap junction channel at 3.5 Å resolution text 65 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-08-01 758 research papers 0907-4449 med@iucr.org 766 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5059 The fitting of high-resolution structures into low-resolution densities obtained from techniques such as electron microscopy or small-angle X-ray scattering can yield powerful new insights. While several algorithms for achieving optimal fits have recently been developed, relatively little effort has been devoted to developing objective measures for judging the quality of the resulting fits, in particular with regard to the danger of overfitting. Here, a general method is presented for obtaining confidence intervals for atomic coordinates resulting from fitting of atomic resolution domain structures into low-resolution densities using well established statistical tools. It is demonstrated that the resulting confidence intervals are sufficiently accurate to allow meaningful statistical tests and to provide tools for detecting potential overfitting. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Volkmann, N. 2009-07-01 doi:10.1107/S0907444909012876 International Union of Crystallography This paper describes procedures for obtaining confidence intervals for coordinate locations resulting from the fitting of atomic models into low-resolution densities. en CONFIDENCE INTERVALS; FITTING TO LOW-RESOLUTION DENSITIES; ELECTRON MICROSCOPY The fitting of high-resolution structures into low-resolution densities obtained from techniques such as electron microscopy or small-angle X-ray scattering can yield powerful new insights. While several algorithms for achieving optimal fits have recently been developed, relatively little effort has been devoted to developing objective measures for judging the quality of the resulting fits, in particular with regard to the danger of overfitting. Here, a general method is presented for obtaining confidence intervals for atomic coordinates resulting from fitting of atomic resolution domain structures into low-resolution densities using well established statistical tools. It is demonstrated that the resulting confidence intervals are sufficiently accurate to allow meaningful statistical tests and to provide tools for detecting potential overfitting. text/html Confidence intervals for fitting of atomic models into low-resolution densities text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 679 research papers 0907-4449 med@iucr.org 689 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5054 An expectation-maximization algorithm for maximum-likelihood refinement of electron-microscopy images is presented that is based on fitting mixtures of multivariate t-distributions. The novel algorithm has intrinsic characteristics for providing robustness against atypical observations in the data, which is illustrated using an experimental test set with artificially generated outliers. Tests on experimental data revealed only minor differences in two-dimensional classifications, while three-dimensional classification with the new algorithm gave stronger elongation factor G density in the corresponding class of a structurally heterogeneous ribosome data set than the conventional algorithm for Gaussian mixtures. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Scheres, S.H.W. Carazo, J.-M. 2009-07-01 doi:10.1107/S0907444909012049 International Union of Crystallography An expectation-maximization algorithm for maximum-likelihood refinement of electron-microscopy data is presented that is based on finite mixtures of multivariate t-distributions. Compared with the conventionally employed Gaussian mixture model, the t-distribution provides robustness against outliers in the data. en ELECTRON MICROSCOPY; MAXIMUM-LIKELIHOOD REFINEMENT; EXPECTATION-MAXIMIZATION ALGORITHM; ROBUSTNESS An expectation-maximization algorithm for maximum-likelihood refinement of electron-microscopy images is presented that is based on fitting mixtures of multivariate t-distributions. The novel algorithm has intrinsic characteristics for providing robustness against atypical observations in the data, which is illustrated using an experimental test set with artificially generated outliers. Tests on experimental data revealed only minor differences in two-dimensional classifications, while three-dimensional classification with the new algorithm gave stronger elongation factor G density in the corresponding class of a structurally heterogeneous ribosome data set than the conventional algorithm for Gaussian mixtures. text/html Introducing robustness to maximum-likelihood refinement of electron-microsopy data text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 672 research papers 0907-4449 med@iucr.org 678 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ea5095 Estimates of the quality of experimental maps are important in many stages of structure determination of macromolecules. Map quality is defined here as the correlation between a map and the corresponding map obtained using phases from the final refined model. Here, ten different measures of experimental map quality were examined using a set of 1359 maps calculated by re-analysis of 246 solved MAD, SAD and MIR data sets. A simple Bayesian approach to estimation of map quality from one or more measures is presented. It was found that a Bayesian estimator based on the skewness of the density values in an electron-density map is the most accurate of the ten individual Bayesian estimators of map quality examined, with a correlation between estimated and actual map quality of 0.90. A combination of the skewness of electron density with the local correlation of r.m.s. density gives a further improvement in estimating map quality, with an overall correlation coefficient of 0.92. The PHENIX AutoSol wizard carries out automated structure solution based on any combination of SAD, MAD, SIR or MIR data sets. The wizard is based on tools from the PHENIX package and uses the Bayesian estimates of map quality described here to choose the highest quality solutions after experimental phasing. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Terwilliger, T.C. Adams, P.D. Read, R.J. McCoy, A.J. Moriarty, N.W. Grosse-Kunstleve, R.W. Afonine, P.V. Zwart, P.H. Hung, L.-W. 2009-06-01 doi:10.1107/S0907444909012098 International Union of Crystallography Ten measures of experimental electron-density-map quality are examined and the skewness of electron density is found to be the best indicator of actual map quality. A Bayesian approach to estimating map quality is developed and used in the PHENIX AutoSol wizard to make decisions during automated structure solution. en STRUCTURE SOLUTION; SCORING; PROTEIN DATA BANK; PHASING; DECISION-MAKING; PHENIX; EXPERIMENTAL ELECTRON-DENSITY MAPS Estimates of the quality of experimental maps are important in many stages of structure determination of macromolecules. Map quality is defined here as the correlation between a map and the corresponding map obtained using phases from the final refined model. Here, ten different measures of experimental map quality were examined using a set of 1359 maps calculated by re-analysis of 246 solved MAD, SAD and MIR data sets. A simple Bayesian approach to estimation of map quality from one or more measures is presented. It was found that a Bayesian estimator based on the skewness of the density values in an electron-density map is the most accurate of the ten individual Bayesian estimators of map quality examined, with a correlation between estimated and actual map quality of 0.90. A combination of the skewness of electron density with the local correlation of r.m.s. density gives a further improvement in estimating map quality, with an overall correlation coefficient of 0.92. The PHENIX AutoSol wizard carries out automated structure solution based on any combination of SAD, MAD, SIR or MIR data sets. The wizard is based on tools from the PHENIX package and uses the Bayesian estimates of map quality described here to choose the highest quality solutions after experimental phasing. text/html Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard text 65 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-06-01 582 research papers 0907-4449 med@iucr.org 601 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5126 Archaeal RadAs are close homologues of eukaryal Rad51s (∼40% sequence identity). These recombinases promote ATP hydrolysis and a hallmark strand-exchange reaction between homologous single-stranded and double-stranded DNA sub­strates. Pairing of the 3′-overhangs located at the damaged DNA with a homologous double-stranded DNA enables the re-synthesis of the damaged region using the homologous DNA as the template. In recent studies, conformational changes in the DNA-interacting regions of Methanococcus voltae RadA have been correlated with the presence of activity-stimulating potassium or calcium ions in the ATPase centre. The series of crystal structures of M. maripaludis RadA presented here further suggest the conservation of an allosteric switch in the ATPase centre which controls the conformational status of DNA-interacting loops. Structural comparison with the distant Escherichia coli RecA homologue supports the notion that the conserved Lys248 and Lys250 residues in RecA play a role similar to that of cations in RadA. The conservation of a cationic bridge between the DNA-interacting L2 region and the terminal phosphate of ATP, together with the apparent stability of the nucleoprotein filament, suggests a gap-displacement model which may explain the advantage of ATP hydrolysis for DNA-strand exchange. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Li, Y. He, Y. Luo, Y. 2009-06-01 doi:10.1107/S0907444909011871 International Union of Crystallography Structural conservation in the ATPase centers of RadA, Rad51 and RecA recombinases suggests conformational switching between high and low-affinity states for DNA in concert with cycles ATP hydrolysis. Such iteration would be advantageous for DNA strand exchange by optimizing the pairing between single-stranded and double-stranded DNA substrates. en RADA; RAD51; RECA; RECOMBINASES; HOMOLOGOUS RECOMBINATION; DNA STRAND EXCHANGE; ATPASES Archaeal RadAs are close homologues of eukaryal Rad51s (∼40% sequence identity). These recombinases promote ATP hydrolysis and a hallmark strand-exchange reaction between homologous single-stranded and double-stranded DNA sub­strates. Pairing of the 3′-overhangs located at the damaged DNA with a homologous double-stranded DNA enables the re-synthesis of the damaged region using the homologous DNA as the template. In recent studies, conformational changes in the DNA-interacting regions of Methanococcus voltae RadA have been correlated with the presence of activity-stimulating potassium or calcium ions in the ATPase centre. The series of crystal structures of M. maripaludis RadA presented here further suggest the conservation of an allosteric switch in the ATPase centre which controls the conformational status of DNA-interacting loops. Structural comparison with the distant Escherichia coli RecA homologue supports the notion that the conserved Lys248 and Lys250 residues in RecA play a role similar to that of cations in RadA. The conservation of a cationic bridge between the DNA-interacting L2 region and the terminal phosphate of ATP, together with the apparent stability of the nucleoprotein filament, suggests a gap-displacement model which may explain the advantage of ATP hydrolysis for DNA-strand exchange. text/html Conservation of a conformational switch in RadA recombinase from Methanococcus maripaludis text 65 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-06-01 602 research papers 0907-4449 med@iucr.org 610 1399-0047 http://scripts.iucr.org/cgi-bin/paper?tm5033 X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallo­graphic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Adams, P.D. Mustyakimov, M. Afonine, P.V. Langan, P. 2009-06-01 doi:10.1107/S0907444909011548 International Union of Crystallography X-ray and neutron crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. en JOINT X-RAY AND NEUTRON CRYSTALLOGRAPHY; STRUCTURE REFINEMENT X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallo­graphic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains. text/html Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules text 65 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-06-01 567 research papers 0907-4449 med@iucr.org 573 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5056 Most known methods for the determination of the structure of macromolecular complexes are limited or at least restricted at some point by their computational demands. Recent developments in information technology such as multicore, parallel and GPU processing can be used to overcome these limitations. In particular, graphics processing units (GPUs), which were originally developed for rendering real-time effects in computer games, are now ubiquitous and provide unprecedented computational power for scientific applications. Each parallel-processing paradigm alone can improve overall performance; the increased computational performance obtained by combining all paradigms, unleashing the full power of today's technology, makes certain applications feasible that were previously virtually impossible. In this article, state-of-the-art paradigms are introduced, the tools and infrastructure needed to apply these paradigms are presented and a state-of-the-art infrastructure and solution strategy for moving scientific applications to the next generation of computer hardware is outlined. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Schmeisser, M. Heisen, B.C. Luettich, M. Busche, B. Hauer, F. Koske, T. Knauber, K.-H. Stark, H. 2009-07-01 doi:10.1107/S0907444909011433 International Union of Crystallography An introduction to the current paradigm shift towards concurrency in software. en PARALLEL PROCESSING; GPU PROCESSING; DISTRIBUTED HETEROGENEOUS COMPUTING; NONDEDICATED SYSTEMS; MULTICORE PERFORMANCE; CLUSTER COMPUTING; ELECTRON MICROSCOPY Most known methods for the determination of the structure of macromolecular complexes are limited or at least restricted at some point by their computational demands. Recent developments in information technology such as multicore, parallel and GPU processing can be used to overcome these limitations. In particular, graphics processing units (GPUs), which were originally developed for rendering real-time effects in computer games, are now ubiquitous and provide unprecedented computational power for scientific applications. Each parallel-processing paradigm alone can improve overall performance; the increased computational performance obtained by combining all paradigms, unleashing the full power of today's technology, makes certain applications feasible that were previously virtually impossible. In this article, state-of-the-art paradigms are introduced, the tools and infrastructure needed to apply these paradigms are presented and a state-of-the-art infrastructure and solution strategy for moving scientific applications to the next generation of computer hardware is outlined. text/html Parallel, distributed and GPU computing technologies in single-particle electron microscopy text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 659 research papers 0907-4449 med@iucr.org 671 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5158 Rotation photographs can be readily indexed if enough candidate Bragg spots are identified to properly sample the reciprocal lattice. However, while automatic indexing algorithms are widely used for macromolecular data processing, they can produce incorrect results in special situations where a subset of Bragg spots is systematically overlooked. This is a potential outcome in cases where a noncrystallographic translational symmetry operator closely mimics an exact crystallo­graphic translation. In these cases, a visual inspection of the diffraction image will reveal alternating strong and weak reflections. However, reliable detection of the weak-intensity reflections by software requires a systematic search for a diffraction signal targeted at specific reciprocal-space locations calculated a priori by considering all possible pseudotranslations. Care must be exercised to distinguish between true lattice diffraction and spurious signals contributed by neighboring overlapping Bragg spots, non-Bragg diffraction and noise. Such procedures have been implemented within the autoindexing program LABELIT and applied to known cases from publicly available data sets. Routine use of this type of signal search adds only a few seconds to the typical run time for autoindexing. The program can be downloaded from http://cci.lbl.gov/labelit. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Sauter, N.K. Zwart, P.H. 2009-06-01 doi:10.1107/S0907444909010725 International Union of Crystallography Lattice patterns containing alternating strong and weak reflections can be identified by a targeted search for the weak signals, permitting a wider range of diffraction patterns to be indexed automatically. en SUBGROUPS; SUBLATTICES; COSETS; NONCRYSTALLOGRAPHIC SYMMETRY Rotation photographs can be readily indexed if enough candidate Bragg spots are identified to properly sample the reciprocal lattice. However, while automatic indexing algorithms are widely used for macromolecular data processing, they can produce incorrect results in special situations where a subset of Bragg spots is systematically overlooked. This is a potential outcome in cases where a noncrystallographic translational symmetry operator closely mimics an exact crystallo­graphic translation. In these cases, a visual inspection of the diffraction image will reveal alternating strong and weak reflections. However, reliable detection of the weak-intensity reflections by software requires a systematic search for a diffraction signal targeted at specific reciprocal-space locations calculated a priori by considering all possible pseudotranslations. Care must be exercised to distinguish between true lattice diffraction and spurious signals contributed by neighboring overlapping Bragg spots, non-Bragg diffraction and noise. Such procedures have been implemented within the autoindexing program LABELIT and applied to known cases from publicly available data sets. Routine use of this type of signal search adds only a few seconds to the typical run time for autoindexing. The program can be downloaded from http://cci.lbl.gov/labelit. text/html Autoindexing the diffraction patterns from crystals with a pseudotranslation text 65 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-06-01 553 research papers 0907-4449 med@iucr.org 559 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5058 The structural refinement of large complexes at the lower resolution limit is often difficult and inefficient owing to the limited number of reflections and the frequently high-level structural flexibility. A new normal-mode-based X-ray crystallo­graphic refinement method has recently been developed that enables anisotropic B-factor refinement using a drastically smaller number of thermal parameters than even isotropic refinement. Here, the method has been systematically tested on a total of eight systems in the resolution range 3.0–3.9 Å. This series of tests established the most applicable scenarios for the method, the detailed procedures for its application and the degree of structural improvement. The results demonstrated substantial model improvement at the lower resolution limit, especially in cases in which other methods such as the translation–libration–screw (TLS) model were not applicable owing to the poorly converged isotropic B-factor distribution. It is expected that this normal-mode-based method will be a useful tool for structural refinement, in particular at the lower resolution limit, in the field of X-ray crystallography. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Ni, F. Poon, B.K. Wang, Q. Ma, J. 2009-07-01 doi:10.1107/S0907444909010695 International Union of Crystallography The application of a new normal-mode-based X-ray crystallographic refinement method to a total of eight structures of moderate resolution is illustrated. en CONFORMATIONAL FLEXIBILITY; ANISOTROPIC THERMAL PARAMETERS; MODERATE RESOLUTION The structural refinement of large complexes at the lower resolution limit is often difficult and inefficient owing to the limited number of reflections and the frequently high-level structural flexibility. A new normal-mode-based X-ray crystallo­graphic refinement method has recently been developed that enables anisotropic B-factor refinement using a drastically smaller number of thermal parameters than even isotropic refinement. Here, the method has been systematically tested on a total of eight systems in the resolution range 3.0–3.9 Å. This series of tests established the most applicable scenarios for the method, the detailed procedures for its application and the degree of structural improvement. The results demonstrated substantial model improvement at the lower resolution limit, especially in cases in which other methods such as the translation–libration–screw (TLS) model were not applicable owing to the poorly converged isotropic B-factor distribution. It is expected that this normal-mode-based method will be a useful tool for structural refinement, in particular at the lower resolution limit, in the field of X-ray crystallography. text/html Application of normal-mode refinement to X-ray crystal structures at the lower resolution limit text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 633 research papers 0907-4449 med@iucr.org 643 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5055 Molecular replacement can fail to find a solution, namely a unique orientation and position of a search model, even when many search models are tested under various conditions. Simultaneous use of the results of these searches may help in the solution of such difficult structures. A closeness between the peaks of several calculated rotation functions may identify the model orientation. The largest and most compact cluster of such peaks usually corresponds to models which are oriented similarly to the molecule under study. A search for the optimal translation may be more problematic and both individual translation functions and straightforward cluster analysis in the space of geometric parameters such as rotation angles and translation vectors may give no result. An improvement may be obtained by performing cluster analysis of the peaks of several translation functions in phase-set space. In this case, the Fourier maps computed using the observed structure-factor magnitudes and the phases calculated from differently positioned models are compared. Again, as a rule, the largest and the most compact cluster corresponds to the correct solution. The result of the updated procedure is no longer a single search model but an averaged Fourier map. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Buehler, A. Urzhumtseva, L. Lunin, V.Y. Urzhumtsev, A. 2009-07-01 doi:10.1107/S090744490900969X International Union of Crystallography Molecular replacement with the simultaneous use of several search functions may solve the phase problem when the conventional molecular-replacement procedure fails to identify the solution. en MOLECULAR REPLACEMENT; PERSISTENT SOLUTION; CLUSTER ANALYSIS; PHASING Molecular replacement can fail to find a solution, namely a unique orientation and position of a search model, even when many search models are tested under various conditions. Simultaneous use of the results of these searches may help in the solution of such difficult structures. A closeness between the peaks of several calculated rotation functions may identify the model orientation. The largest and most compact cluster of such peaks usually corresponds to models which are oriented similarly to the molecule under study. A search for the optimal translation may be more problematic and both individual translation functions and straightforward cluster analysis in the space of geometric parameters such as rotation angles and translation vectors may give no result. An improvement may be obtained by performing cluster analysis of the peaks of several translation functions in phase-set space. In this case, the Fourier maps computed using the observed structure-factor magnitudes and the phases calculated from differently positioned models are compared. Again, as a rule, the largest and the most compact cluster corresponds to the correct solution. The result of the updated procedure is no longer a single search model but an averaged Fourier map. text/html Cluster analysis for phasing with molecular replacement: a feasibility study text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 644 research papers 0907-4449 med@iucr.org 650 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5123 Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloram­phenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-­acyl group. It is concluded that modifications of the 3-­hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Pettigrew, D.M. Roversi, P. Davies, S.G. Russell, A.J. Lea, S.M. 2009-06-01 doi:10.1107/S0907444909005113 International Union of Crystallography The structures of two Dr adhesin (DraE) complexes with chloramphenicol derivatives, namely chloramphenicol succinate and bromamphenicol, have been solved. The structures reveal important functional groups for small-molecule binding and imply possible modifications to the molecule that would permit a more wide-ranging interaction without the toxic side effects associated with chloramphenicol. en DR ADHESINS; DR HAEMAGGLUTININ; DRAE; CHLORAMPHENICOL Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloram­phenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-­acyl group. It is concluded that modifications of the 3-­hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM. text/html A structural study of the interaction between the Dr haemagglutinin DraE and derivatives of chloramphenicol text 65 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-06-01 513 research papers 0907-4449 med@iucr.org 522 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5020 Corrections are made to the article by Kato et al. [Acta Cryst. (2008). D64, 525–531]. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kato, K. Tanaka, H. Sumizawa, T. Yoshimura, M. Yamashita, E. Iwasaki, K. Tsukihara, T. 2009-03-01 doi:10.1107/S0907444909005824 International Union of Crystallography A corrigendum to the article by Kato et al. [Acta Cryst. (2008). D64, 525–531]. en VAULT; RIBONUCLEOPROTEINS; CORRIGENDUM Corrections are made to the article by Kato et al. [Acta Cryst. (2008). D64, 525–531]. text/html A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry. Corrigendum text 65 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-03-01 301 addenda and errata 0907-4449 med@iucr.org 301 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ic5057 Electron microscopy of a macromolecular structure can lead to three-dimensional reconstructions with resolutions that are typically in the 30–10 Å range and sometimes even beyond 10 Å. Fitting atomic models of the individual components of the macromolecular structure (e.g. those obtained by X-ray crystallo­graphy or nuclear magnetic resonance) into an electron-microscopy map allows the interpretation of the latter at near-atomic resolution, providing insight into the interactions between the components. Graphical software is presented that was designed for the interactive fitting and refinement of atomic models into electron-microscopy reconstructions. Several characteristics enable it to be applied over a wide range of cases and resolutions. Firstly, calculations are performed in reciprocal space, which results in fast algorithms. This allows the entire reconstruction (or at least a sizeable portion of it) to be used by taking into account the symmetry of the reconstruction both in the calculations and in the graphical display. Secondly, atomic models can be placed graphically in the map while the correlation between the model-based electron density and the electron-microscopy reconstruction is computed and displayed in real time. The positions and orientations of the models are refined by a least-squares minimization. Thirdly, normal-mode calculations can be used to simulate conformational changes between the atomic model of an individual component and its corresponding density within a macromolecular complex determined by electron microscopy. These features are illustrated using three practical cases with different symmetries and resolutions. The software, together with examples and user instructions, is available free of charge at http://mem.ibs.fr/UROX/. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Siebert, X. Navaza, J. 2009-07-01 doi:10.1107/S0907444909008671 International Union of Crystallography UROX is software designed for the interactive fitting of atomic models into electron-microscopy reconstructions. The main features of the software are presented, along with a few examples. en UROX; ELECTRON MICROSCOPY; MODEL FITTING; GRAPHICAL SOFTWARE; NORMAL MODES Electron microscopy of a macromolecular structure can lead to three-dimensional reconstructions with resolutions that are typically in the 30–10 Å range and sometimes even beyond 10 Å. Fitting atomic models of the individual components of the macromolecular structure (e.g. those obtained by X-ray crystallo­graphy or nuclear magnetic resonance) into an electron-microscopy map allows the interpretation of the latter at near-atomic resolution, providing insight into the interactions between the components. Graphical software is presented that was designed for the interactive fitting and refinement of atomic models into electron-microscopy reconstructions. Several characteristics enable it to be applied over a wide range of cases and resolutions. Firstly, calculations are performed in reciprocal space, which results in fast algorithms. This allows the entire reconstruction (or at least a sizeable portion of it) to be used by taking into account the symmetry of the reconstruction both in the calculations and in the graphical display. Secondly, atomic models can be placed graphically in the map while the correlation between the model-based electron density and the electron-microscopy reconstruction is computed and displayed in real time. The positions and orientations of the models are refined by a least-squares minimization. Thirdly, normal-mode calculations can be used to simulate conformational changes between the atomic model of an individual component and its corresponding density within a macromolecular complex determined by electron microscopy. These features are illustrated using three practical cases with different symmetries and resolutions. The software, together with examples and user instructions, is available free of charge at http://mem.ibs.fr/UROX/. text/html UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 651 research papers 0907-4449 med@iucr.org 658 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5143 Unit-cell determination is the first step towards the structure solution of an unknown crystal form. Standard procedures for unit-cell determination cannot cope with data collections that consist of single diffraction patterns of multiple crystals, each with an unknown orientation. However, for beam-sensitive nanocrystals these are often the only data that can be obtained. An algorithm for unit-cell determination that uses randomly oriented electron-diffraction patterns with unknown angular relationships is presented here. The algorithm determined the unit cells of mineral, pharmaceutical and protein nanocrystals in orthorhombic high- and low-symmetry space groups, allowing (well oriented) patterns to be indexed. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Jiang, L. Georgieva, D. Zandbergen, H.W. Abrahams, J.P. 2009-07-01 doi:10.1107/S0907444909003163 International Union of Crystallography An algorithm is described that calculates the most likely primitive unit cell given a set of randomly oriented electron-diffraction patterns with unknown angular relationships. en ELECTRON DIFFRACTION; NANOCRYSTALS; UNIT-CELL DETERMINATION; UNKNOWN ORIENTATION Unit-cell determination is the first step towards the structure solution of an unknown crystal form. Standard procedures for unit-cell determination cannot cope with data collections that consist of single diffraction patterns of multiple crystals, each with an unknown orientation. However, for beam-sensitive nanocrystals these are often the only data that can be obtained. An algorithm for unit-cell determination that uses randomly oriented electron-diffraction patterns with unknown angular relationships is presented here. The algorithm determined the unit cells of mineral, pharmaceutical and protein nanocrystals in orthorhombic high- and low-symmetry space groups, allowing (well oriented) patterns to be indexed. text/html Unit-cell determination from randomly oriented electron-diffraction patterns text 65 7 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2009-07-01 625 research papers 0907-4449 med@iucr.org 632 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5144 The discovery of ligands that bind specifically to a targeted protein benefits from the development of generic assays for high-throughput screening of a library of chemicals. Protein powder diffraction (PPD) has been proposed as a potential method for use as a structure-based assay for high-throughput screening applications. Building on this effort, powder samples of bound/unbound states of soluble hen-egg white lysozyme precipitated with sodium chloride were compared. The correlation coefficients calculated between the raw diffraction profiles were consistent with the known binding properties of the ligands and suggested that the PPD approach can be used even prior to a full description using stereochemically restrained Rietveld refinement. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Allaire, M. Moiseeva, N. Botez, C.E. Engel, M.A. Stephens, P.W. 2009-04-01 doi:10.1107/S090744490900256X International Union of Crystallography The correlation coefficients calculated between raw powder diffraction profiles can be used to identify ligand-bound/unbound states of lysozyme. en PROTEIN-LIGAND COMPLEXES; ASSAYS; POWDER DIFFRACTION; LYSOZYME The discovery of ligands that bind specifically to a targeted protein benefits from the development of generic assays for high-throughput screening of a library of chemicals. Protein powder diffraction (PPD) has been proposed as a potential method for use as a structure-based assay for high-throughput screening applications. Building on this effort, powder samples of bound/unbound states of soluble hen-egg white lysozyme precipitated with sodium chloride were compared. The correlation coefficients calculated between the raw diffraction profiles were consistent with the known binding properties of the ligands and suggested that the PPD approach can be used even prior to a full description using stereochemically restrained Rietveld refinement. text/html On the possibility of using polycrystalline material in the development of structure-based generic assays text 65 4 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-04-01 379 short communications 0907-4449 med@iucr.org 382 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5128 Knowledge of the three-dimensional structures of the carbo­hydrate molecules is indispensable for a full understanding of the molecular processes in which carbohydrates are involved, such as protein glycosylation or protein–carbohydrate interactions. The Protein Data Bank (PDB) is a valuable resource for three-dimensional structural information on glycoproteins and protein–carbohydrate complexes. Unfortunately, many carbohydrate moieties in the PDB contain inconsistencies or errors. This article gives an overview of the information that can be obtained from individual PDB entries and from statistical analyses of sets of three-dimensional structures, of typical problems that arise during the analysis of carbohydrate three-dimensional structures and of the validation tools that are currently available to scientists to evaluate the quality of these structures. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Lütteke, T. 2009-02-01 doi:10.1107/S0907444909001905 International Union of Crystallography The article summarizes the information that is gained from and the errors that are found in carbohydrate structures in the Protein Data Bank. Validation tools that can locate these errors are described. en VALIDATION; GLYCOSYLATION; GLYCAN CONFORMATION; THREE-DIMENSIONAL STRUCTURE QUALITY Knowledge of the three-dimensional structures of the carbo­hydrate molecules is indispensable for a full understanding of the molecular processes in which carbohydrates are involved, such as protein glycosylation or protein–carbohydrate interactions. The Protein Data Bank (PDB) is a valuable resource for three-dimensional structural information on glycoproteins and protein–carbohydrate complexes. Unfortunately, many carbohydrate moieties in the PDB contain inconsistencies or errors. This article gives an overview of the information that can be obtained from individual PDB entries and from statistical analyses of sets of three-dimensional structures, of typical problems that arise during the analysis of carbohydrate three-dimensional structures and of the validation tools that are currently available to scientists to evaluate the quality of these structures. text/html Analysis and validation of carbohydrate three-dimensional structures text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 156 research papers 0907-4449 med@iucr.org 168 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5129 The determination of incorrect structures for the ABC transporter MsbA gave rise to questions of how this could have occurred. Methodological aspects of the MsbA structure determination are explored in light of this error. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Jeffrey, P.D. 2009-02-01 doi:10.1107/S0907444909001292 International Union of Crystallography An analysis is presented of the methodological errors that led to the incorrect structure of MsbA. en PROTEIN CRYSTALLOGRAPHY; METHODOLOGY The determination of incorrect structures for the ABC transporter MsbA gave rise to questions of how this could have occurred. Methodological aspects of the MsbA structure determination are explored in light of this error. text/html Analysis of errors in the structure determination of MsbA text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 193 research papers 0907-4449 med@iucr.org 199 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5123 Limitations to the data and subjectivity in the structure-determination process may cause errors in macromolecular crystal structures. Appropriate validation techniques may be used to reveal problems in structures, ideally before they are analysed, published or deposited. Additionally, such tech­niques may be used a posteriori to assess the (relative) merits of a model by potential users. Weak validation methods and statistics assess how well a model reproduces the information that was used in its construction (i.e. experimental data and prior knowledge). Strong methods and statistics, on the other hand, test how well a model predicts data or information that were not used in the structure-determination process. These may be data that were excluded from the process on purpose, general knowledge about macromolecular structure, information about the biological role and biochemical activity of the molecule under study or its mutants or complexes and predictions that are based on the model and that can be tested experimentally. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Kleywegt, G.J. 2009-02-01 doi:10.1107/S090744490900081X International Union of Crystallography The need for validation of macromolecular crystal structures is discussed. A general approach to validation is presented, together with examples of its implementation in the special case of macromolecular crystallography. en VALIDATION Limitations to the data and subjectivity in the structure-determination process may cause errors in macromolecular crystal structures. Appropriate validation techniques may be used to reveal problems in structures, ideally before they are analysed, published or deposited. Additionally, such tech­niques may be used a posteriori to assess the (relative) merits of a model by potential users. Weak validation methods and statistics assess how well a model reproduces the information that was used in its construction (i.e. experimental data and prior knowledge). Strong methods and statistics, on the other hand, test how well a model predicts data or information that were not used in the structure-determination process. These may be data that were excluded from the process on purpose, general knowledge about macromolecular structure, information about the biological role and biochemical activity of the molecule under study or its mutants or complexes and predictions that are based on the model and that can be tested experimentally. text/html On vital aid: the why, what and how of validation text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 134 research papers 0907-4449 med@iucr.org 139 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5132 The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Jenni, S. Ban, N. 2009-02-01 doi:10.1107/S0907444909000778 International Union of Crystallography A case of imperfect pseudo-merohedral twinning in monoclinic crystals of fungal fatty acid synthase is discussed. A space-group transition during crystal dehydration resulted in a Moiré pattern-like interference of the twinned diffraction patterns. en IMPERFECT PSEUDO-MEROHEDRAL TWINNING; FUNGAL FATTY ACID SYNTHASE The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model. text/html Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthase text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 101 research papers 0907-4449 med@iucr.org 111 1399-0047 http://scripts.iucr.org/cgi-bin/paper?lv5003 A crystallographic macromolecular model is typically characterized by a list of quality criteria, such as R factors, deviations from ideal stereochemistry and average B factors, which are usually provided as tables in publications or in structural databases. In order to facilitate a quick model-quality evaluation, a graphical representation is proposed. Each key parameter such as R factor or bond-length deviation from `ideal values' is shown graphically as a point on a `ruler'. These rulers are plotted as a set of lines with the same origin, forming a hub and spokes. Different parts of the rulers are coloured differently to reflect the frequency (red for a low frequency, blue for a high frequency) with which the corresponding values are observed in a reference set of structures determined previously. The points for a given model marked on these lines are connected to form a polygon. A polygon that is strongly compressed or dilated along some axes reveals unusually low or high values of the corresponding characteristics. Polygon vertices in `red zones' indicate parameters which lie outside typical values. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Urzhumtseva, L. Afonine, P.V. Adams, P.D. Urzhumtsev, A. 2009-03-01 doi:10.1107/S0907444908044296 International Union of Crystallography The representation of crystallographic model characteristics in the form of a polygon allows the quick comparison of a model with a set of previously solved structures. en MODEL QUALITY; PDB; VALIDATION; REFINEMENT; PHENIX A crystallographic macromolecular model is typically characterized by a list of quality criteria, such as R factors, deviations from ideal stereochemistry and average B factors, which are usually provided as tables in publications or in structural databases. In order to facilitate a quick model-quality evaluation, a graphical representation is proposed. Each key parameter such as R factor or bond-length deviation from `ideal values' is shown graphically as a point on a `ruler'. These rulers are plotted as a set of lines with the same origin, forming a hub and spokes. Different parts of the rulers are coloured differently to reflect the frequency (red for a low frequency, blue for a high frequency) with which the corresponding values are observed in a reference set of structures determined previously. The points for a given model marked on these lines are connected to form a polygon. A polygon that is strongly compressed or dilated along some axes reveals unusually low or high values of the corresponding characteristics. Polygon vertices in `red zones' indicate parameters which lie outside typical values. text/html Crystallographic model quality at a glance text 65 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-03-01 297 short communications 0907-4449 med@iucr.org 300 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hm5071 Death-associated protein kinase (DAPK) is a member of the Ca2+/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg2+ at 1.85 Å resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK–ADP–Mg2+ was compared with a newly determined DAPK–AMP-PNP–Mg2+ structure and the previously determined apo DAPK structure (PDB code 1jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 McNamara, L.K. Watterson, D.M. Brunzelle, J.S. 2009-03-01 doi:10.1107/S0907444908043679 International Union of Crystallography The crystal structures of DAPK–ADP–Mg2+ and DAPK–AMP-PNP–Mg2+ complexes were determined at 1.85 and 2.00 Å resolution, respectively. Comparison of the two nucleotide-bound states with apo DAPK revealed localized changes in the glycine-rich loop region that were indicative of a transition from a more open state to a more closed state on binding of the nucleotide substrate and to an intermediate state with the bound nucleotide product. en DEATH-ASSOCIATED PROTEIN KINASE; SERINE/THREONINE PROTEIN KINASES; NUCLEOTIDES; GLYCINE-RICH LOOPS; CA2+/CALMODULIN-REGULATED PROTEIN KINASES; APOPTOSIS; BRAIN Death-associated protein kinase (DAPK) is a member of the Ca2+/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg2+ at 1.85 Å resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK–ADP–Mg2+ was compared with a newly determined DAPK–AMP-PNP–Mg2+ structure and the previously determined apo DAPK structure (PDB code 1jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK. text/html Structural insight into nucleotide recognition by human death-associated protein kinase text 65 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-03-01 241 research papers 0907-4449 med@iucr.org 248 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5131 As an example of structure determination in the 3.5–4.5 Å resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3 Å resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at ∼4 Å resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Brunger, A.T. DeLaBarre, B. Davies, J.M. Weis, W.I. 2009-02-01 doi:10.1107/S0907444908043795 International Union of Crystallography Refinement is meaningful even at 4 Å or lower, but with present methodologies it should start from high-resolution crystal structures whenever possible. en REFINEMENT; LOW RESOLUTION; STRUCTURE VALIDATION; ATPASES; P97/VCP As an example of structure determination in the 3.5–4.5 Å resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3 Å resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at ∼4 Å resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible. text/html X-ray structure determination at low resolution text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 128 research papers 0907-4449 med@iucr.org 133 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5124 Automated structure validation was introduced in chemical crystallography about 12 years ago as a tool to assist practitioners with the exponential growth in crystal structure analyses. Validation has since evolved into an easy-to-use checkCIF/PLATON web-based IUCr service. The result of a crystal structure determination has to be supplied as a CIF-formatted computer-readable file. The checking software tests the data in the CIF for completeness, quality and consistency. In addition, the reported structure is checked for incomplete analysis, errors in the analysis and relevant issues to be verified. A validation report is generated in the form of a list of ALERTS on the issues to be corrected, checked or commented on. Structure validation has largely eliminated obvious problems with structure reports published in IUCr journals, such as refinement in a space group of too low symmetry. This paper reports on the current status of structure validation and possible future extensions. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Spek, A.L. 2009-02-01 doi:10.1107/S090744490804362X International Union of Crystallography This paper reports on the current status of structure validation in chemical crystallography. en VALIDATION; CHECKCIF; PLATON Automated structure validation was introduced in chemical crystallography about 12 years ago as a tool to assist practitioners with the exponential growth in crystal structure analyses. Validation has since evolved into an easy-to-use checkCIF/PLATON web-based IUCr service. The result of a crystal structure determination has to be supplied as a CIF-formatted computer-readable file. The checking software tests the data in the CIF for completeness, quality and consistency. In addition, the reported structure is checked for incomplete analysis, errors in the analysis and relevant issues to be verified. A validation report is generated in the form of a list of ALERTS on the issues to be corrected, checked or commented on. Structure validation has largely eliminated obvious problems with structure reports published in IUCr journals, such as refinement in a space group of too low symmetry. This paper reports on the current status of structure validation and possible future extensions. text/html Structure validation in chemical crystallography text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 148 research papers 0907-4449 med@iucr.org 155 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5117 A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Trinh, C.H. Asipu, A. Bonthron, D.T. Phillips, S.E.V. 2009-03-01 doi:10.1107/S0907444908041115 International Union of Crystallography The structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and peripheral KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP have been solved. The differences between KHK-A and KHK-C resulting from the spliced region are subtle and affect thermostability and probably flexibility; the mutations causing fructosuria were modelled. en KETOHEXOKINASE; ALTERNATIVELY SPLICED ISOFORMS; FRUCTOKINASE; FRUCTOSURIA A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure. text/html Structures of alternatively spliced isoforms of human ketohexokinase text 65 3 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-03-01 201 research papers 0907-4449 med@iucr.org 211 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5130 Although many factors influence the quality of a macromolecular crystal structure, validation criteria are usually only calibrated using one of these factors, the resolution. For many purposes this is sufficient, but there are times when one wishes to compare one set of structures with another and the comparison may be invalidated by systematic differences between the sets in factors other than resolution. This problem can be circumvented by borrowing from medicine the idea of the case-matched control: each structure of interest is matched with a control structure that has similar values for all relevant factors considered in this study. In addition to resolution, these include the size of the structure (as measured by the volume of the asymmetric unit) and the year of deposition. This approach has been applied to address two questions: whether structures from structural genomics efforts reach the same level of quality as structures from traditional sources and whether the impact factor of the journal in which a structure is published correlates with structure quality. In both cases, once factors influencing quality have been controlled in the comparison, there is little evidence for a systematic difference in quality. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Read, R.J. Kleywegt, G.J. 2009-02-01 doi:10.1107/S0907444908041085 International Union of Crystallography A case-matched control protocol provides a useful method to compare sets of macromolecular structures. en PROTEIN STRUCTURES; VALIDATION; CASE-CONTROL METHOD; RESOLUTION; STRUCTURAL GENOMICS Although many factors influence the quality of a macromolecular crystal structure, validation criteria are usually only calibrated using one of these factors, the resolution. For many purposes this is sufficient, but there are times when one wishes to compare one set of structures with another and the comparison may be invalidated by systematic differences between the sets in factors other than resolution. This problem can be circumvented by borrowing from medicine the idea of the case-matched control: each structure of interest is matched with a control structure that has similar values for all relevant factors considered in this study. In addition to resolution, these include the size of the structure (as measured by the volume of the asymmetric unit) and the year of deposition. This approach has been applied to address two questions: whether structures from structural genomics efforts reach the same level of quality as structures from traditional sources and whether the impact factor of the journal in which a structure is published correlates with structure quality. In both cases, once factors influencing quality have been controlled in the comparison, there is little evidence for a systematic difference in quality. text/html Case-controlled structure validation text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 140 research papers 0907-4449 med@iucr.org 147 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5126 The interpretation of low-resolution X-ray crystallographic data proves to be challenging even for the most experienced crystallographer. Ambiguity in the electron-density map makes main-chain tracing and side-chain assignment difficult. However, the number of structures solved at resolutions poorer than 3.5 Å is growing rapidly and the structures are often of high biological interest and importance. Here, the challenges faced in electron-density interpretation, the strategies that have been employed to overcome them and developments to automate the process are reviewed. The methods employed in model generation from electron microscopy, which share many of the same challenges in providing high-confidence models of macromolecular structures and assemblies, are also considered. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Karmali, A.M. Blundell, T.L. Furnham, N. 2009-02-01 doi:10.1107/S0907444908040006 International Union of Crystallography Interpretation of low-resolution X-ray crystallographic data can prove to be a difficult task. The challenges faced in electron-density interpretation, the strategies that have been employed to overcome them and developments to automate the process are reviewed. en MODEL BUILDING; LOW-RESOLUTION DATA The interpretation of low-resolution X-ray crystallographic data proves to be challenging even for the most experienced crystallographer. Ambiguity in the electron-density map makes main-chain tracing and side-chain assignment difficult. However, the number of structures solved at resolutions poorer than 3.5 Å is growing rapidly and the structures are often of high biological interest and importance. Here, the challenges faced in electron-density interpretation, the strategies that have been employed to overcome them and developments to automate the process are reviewed. The methods employed in model generation from electron microscopy, which share many of the same challenges in providing high-confidence models of macromolecular structures and assemblies, are also considered. text/html Model-building strategies for low-resolution X-ray crystallographic data text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 121 research papers 0907-4449 med@iucr.org 127 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5127 RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin α-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Brueckner, F. Armache, K.-J. Cheung, A. Damsma, G.E. Kettenberger, H. Lehmann, E. Sydow, J. Cramer, P. 2009-02-01 doi:10.1107/S0907444908039875 International Union of Crystallography X-ray crystallographic and complementary functional studies have contributed significantly to the current understanding of gene transcription. Here, recent structure–function studies on various aspects of the elongation phase of transcription are summarized. en NUCLEIC ACIDS; TRANSCRIPTION ELONGATION; RNA POLYMERASE II; NUCLEOTIDE-ADDITION CYCLE; TRANSLOCATION; TRANSCRIPTION FACTOR IIS; DNA-DAMAGE RECOGNITION; RNA-DEPENDENT RNA POLYMERASE ACTIVITY RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin α-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template. text/html Structure–function studies of the RNA polymerase II elongation complex text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 112 research papers 0907-4449 med@iucr.org 120 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5125 The deposition of X-ray data along with the customary structural models defining PDB entries makes it possible to apply large-scale re-refinement protocols to these entries, thus giving users the benefit of improvements in X-ray methods that have occurred since the structure was deposited. Auto­mated gradient refinement is an effective method to achieve this goal, but real-space intervention is most often required in order to adequately address problems detected by structure-validation software. In order to improve the existing protocol, automated re-refinement was combined with structure validation and difference-density peak analysis to produce a catalogue of problems in PDB entries that are amenable to automatic correction. It is shown that re-refinement can be effective in producing improvements, which are often associated with the systematic use of the TLS parameterization of B factors, even for relatively new and high-resolution PDB entries, while the accompanying manual or semi-manual map analysis and fitting steps show good prospects for eventual automation. It is proposed that the potential for simultaneous improvements in methods and in re-refinement results be further encouraged by broadening the scope of depositions to include refinement metadata and ultimately primary rather than reduced X-ray data. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Joosten, R.P. Womack, T. Vriend, G. Bricogne, G. 2009-02-01 doi:10.1107/S0907444908037591 International Union of Crystallography An evaluation of validation and real-space intervention possibilities for improving existing automated (re-)refinement methods. en RE-REFINEMENT The deposition of X-ray data along with the customary structural models defining PDB entries makes it possible to apply large-scale re-refinement protocols to these entries, thus giving users the benefit of improvements in X-ray methods that have occurred since the structure was deposited. Auto­mated gradient refinement is an effective method to achieve this goal, but real-space intervention is most often required in order to adequately address problems detected by structure-validation software. In order to improve the existing protocol, automated re-refinement was combined with structure validation and difference-density peak analysis to produce a catalogue of problems in PDB entries that are amenable to automatic correction. It is shown that re-refinement can be effective in producing improvements, which are often associated with the systematic use of the TLS parameterization of B factors, even for relatively new and high-resolution PDB entries, while the accompanying manual or semi-manual map analysis and fitting steps show good prospects for eventual automation. It is proposed that the potential for simultaneous improvements in methods and in re-refinement results be further encouraged by broadening the scope of depositions to include refinement metadata and ultimately primary rather than reduced X-ray data. text/html Re-refinement from deposited X-ray data can deliver improved models for most PDB entries text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 176 research papers 0907-4449 med@iucr.org 185 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5118 The archetypical member of the small multidrug-resistance family is EmrE, a multidrug transporter that extrudes toxic polyaromatic cations from the cell coupled to the inward movement of protons down a concentration gradient. The architecture of EmrE was first defined from the analysis of two-dimensional crystals by cryoelectron microscopy (cryo-EM), which showed that EmrE was an unusual asymmetric dimer formed from a bundle of eight α-helices. The most favoured interpretation of the structure was that the monomers were oriented in opposite orientations in the membrane in an antiparallel orientation. A model was subsequently built based upon the cryo-EM data and evolutionary constraints and this model was consistent with mutagenic data indicating which amino-acid residues were important for substrate binding and transport. Two X-ray structures that differed significantly from the cryo-EM structure were subsequently retracted owing to a data-analysis error. However, the revised X-ray structure with substrate bound is extremely similar to the model built from the cryo-EM structure (r.m.s.d. of 1.4 Å), suggesting that the proposed antiparallel orientation of the monomers is indeed correct; this represents a new structural paradigm in membrane-protein structures. The vast majority of mutagenic and biochemical data corroborate this structure, although cross-linking studies and recent EPR data apparently support a model of EmrE that contains parallel dimers. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Korkhov, V.M. Tate, C.G. 2009-02-01 doi:10.1107/S0907444908036640 International Union of Crystallography The ongoing story of the structure of EmrE, the archetypical member of the small multidrug-resistance family, is described. en EMRE; MULTIDRUG TRANSPORTERS; SMALL MULTIDRUG-RESISTANCE FAMILY The archetypical member of the small multidrug-resistance family is EmrE, a multidrug transporter that extrudes toxic polyaromatic cations from the cell coupled to the inward movement of protons down a concentration gradient. The architecture of EmrE was first defined from the analysis of two-dimensional crystals by cryoelectron microscopy (cryo-EM), which showed that EmrE was an unusual asymmetric dimer formed from a bundle of eight α-helices. The most favoured interpretation of the structure was that the monomers were oriented in opposite orientations in the membrane in an antiparallel orientation. A model was subsequently built based upon the cryo-EM data and evolutionary constraints and this model was consistent with mutagenic data indicating which amino-acid residues were important for substrate binding and transport. Two X-ray structures that differed significantly from the cryo-EM structure were subsequently retracted owing to a data-analysis error. However, the revised X-ray structure with substrate bound is extremely similar to the model built from the cryo-EM structure (r.m.s.d. of 1.4 Å), suggesting that the proposed antiparallel orientation of the monomers is indeed correct; this represents a new structural paradigm in membrane-protein structures. The vast majority of mutagenic and biochemical data corroborate this structure, although cross-linking studies and recent EPR data apparently support a model of EmrE that contains parallel dimers. text/html An emerging consensus for the structure of EmrE text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 186 research papers 0907-4449 med@iucr.org 192 1399-0047 http://scripts.iucr.org/cgi-bin/paper?yt5007 The ability of computers to learn from and annotate large databases of crystallization-trial images provides not only the ability to reduce the workload of crystallization studies, but also an opportunity to annotate crystallization trials as part of a framework for improving screening methods. Here, a system is presented that scores sets of images based on the likelihood of containing crystalline material as perceived by a machine-learning algorithm. The system can be incorporated into existing crystallization-analysis pipelines, whereby specialists examine images as they normally would with the exception that the images appear in rank order according to a simple real-valued score. Promising results are shown for 319 112 images associated with 150 structures solved by the Joint Center for Structural Genomics pipeline during the 2006–2007 year. Overall, the algorithm achieves a mean receiver opera­ting characteristic score of 0.919 and a 78% reduction in human effort per set when considering an absolute score cutoff for screening images, while incurring a loss of five out of 150 structures. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Liu, R. Freund, Y. Spraggon, G. 2008-12-01 doi:10.1107/S090744490802982X International Union of Crystallography A system for scoring images based on the likelihood of containing crystalline material is described. A simulation carried out on a real set of crystallization images demonstrates the utility of such a system in high-throughput environments by substantially reducing the manual workload necessary to detect crystals for X-ray screening. en IMAGE ANALYSIS; MACHINE LEARNING; STRUCTURAL GENOMICS; FEATURE EXTRACTION The ability of computers to learn from and annotate large databases of crystallization-trial images provides not only the ability to reduce the workload of crystallization studies, but also an opportunity to annotate crystallization trials as part of a framework for improving screening methods. Here, a system is presented that scores sets of images based on the likelihood of containing crystalline material as perceived by a machine-learning algorithm. The system can be incorporated into existing crystallization-analysis pipelines, whereby specialists examine images as they normally would with the exception that the images appear in rank order according to a simple real-valued score. Promising results are shown for 319 112 images associated with 150 structures solved by the Joint Center for Structural Genomics pipeline during the 2006–2007 year. Overall, the algorithm achieves a mean receiver opera­ting characteristic score of 0.919 and a 78% reduction in human effort per set when considering an absolute score cutoff for screening images, while incurring a loss of five out of 150 structures. text/html Image-based crystal detection: a machine-learning approach text 64 12 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-12-01 1187 research papers 0907-4449 med@iucr.org 1195 1399-0047 http://scripts.iucr.org/cgi-bin/paper?bw5254 The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ∼10–20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one com­prehensive `hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Gerdts, C.J. Elliott, M. Lovell, S. Mixon, M.B. Napuli, A.J. Staker, B.L. Nollert, P. Stewart, L. 2008-11-01 doi:10.1107/S0907444908028060 International Union of Crystallography The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase. en PROTEIN CRYSTALLIZATION; MICROCAPILLARY PROTEIN CRYSTALLIZATION SYSTEM The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ∼10–20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one com­prehensive `hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants. text/html The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS) text 64 11 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-11-01 1116 research papers 0907-4449 med@iucr.org 1122 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5105 Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Stec, B. Stieglitz, K.A. 2008-09-01 doi:10.1107/S0907444908022002 International Union of Crystallography Comment on Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis by Widboom et al. (2007), Nature (London), 447, 342–345. en LETTERS TO THE EDITOR; COFACTOR-INDEPENDENT DIOXYGENATION; VANCOMYCIN; LATTICE-TRANSLOCATION DEFECT text/html Not so clear on oxygen. Comment on Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis by Widboom et al. (2007), Nature (London), 447, 342–345 text 64 9 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-09-01 1000 letters to the editor 0907-4449 med@iucr.org 1002 1399-0047 http://scripts.iucr.org/cgi-bin/paper?cy5047 Protein crystallization has been revolutionized by the intro­duction of high-throughput technologies, which have led to a speeding up of the process while simultaneously reducing the amount of protein sample necessary. Nonetheless, the chemistry dimension of protein crystallization has remained relatively undeveloped. Most crystallization screens are based on the same set of precipitants. To address this shortcoming, the development of new protein precipitants based on poly-γ-­glutamic acid (PGA) polymers with different molecular-weight ranges is reported here: PGA-LM (low molecular weight) of ∼400 kDa and PGA-HM (high molecular weight) of >1000 kDa. It is also demonstrated that protein precipitants can be expanded further to polymers with much higher molecular weight than those that are currently in use. Furthermore, the modification of PGA-like polymers by covalent attachments of glucosamine substantially improved their solubility without affecting their crystallization properties. Some preliminary PGA-based screens are presented here. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Hu, T.-C. Korczyńska, J. Smith, D.K. Brzozowski, A.M. 2008-09-01 doi:10.1107/S0907444908021616 International Union of Crystallography High-molecular-weight poly-γ-glutamic acid-based polymers have been synthesized, tested and adopted for protein crystallization. en PROTEIN CRYSTALLIZATION; CRYSTALLIZATION PRECIPITANTS; CRYSTALLIZATION SCREENS; HIGH THROUGHPUT Protein crystallization has been revolutionized by the intro­duction of high-throughput technologies, which have led to a speeding up of the process while simultaneously reducing the amount of protein sample necessary. Nonetheless, the chemistry dimension of protein crystallization has remained relatively undeveloped. Most crystallization screens are based on the same set of precipitants. To address this shortcoming, the development of new protein precipitants based on poly-γ-­glutamic acid (PGA) polymers with different molecular-weight ranges is reported here: PGA-LM (low molecular weight) of ∼400 kDa and PGA-HM (high molecular weight) of >1000 kDa. It is also demonstrated that protein precipitants can be expanded further to polymers with much higher molecular weight than those that are currently in use. Furthermore, the modification of PGA-like polymers by covalent attachments of glucosamine substantially improved their solubility without affecting their crystallization properties. Some preliminary PGA-based screens are presented here. text/html High-molecular-weight polymers for protein crystallization: poly-γ-glutamic acid-based precipitants text 64 9 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-09-01 957 research papers 0907-4449 med@iucr.org 963 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5122 The prospect of phasing diffraction data sets `de novo' for proteins with previously unseen folds is appealing but largely untested. In a first systematic exploration of phasing with Rosetta de novo models, it is shown that all-atom refinement of coarse-grained models significantly improves both the model quality and performance in molecular replacement with the Phaser software. 15 new cases of diffraction data sets that are unambiguously phased with de novo models are presented. These diffraction data sets represent nine space groups and span a large range of solvent contents (33–79%) and asymmetric unit copy numbers (1–4). No correlation is observed between the ease of phasing and the solvent content or asymmetric unit copy number. Instead, a weak correlation is found with the length of the modeled protein: larger proteins required somewhat less accurate models to give successful molecular replacement. Overall, the results of this survey suggest that de novo models can phase diffraction data for approximately one sixth of proteins with sizes of 100 residues or less. However, for many of these cases, `de novo phasing with de novo models' requires significant investment of computational power, much greater than 103 CPU days per target. Improvements in conformational search methods will be necessary if molecular replacement with de novo models is to become a practical tool for targets without homology to previously solved protein structures. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Das, R. Baker, D. 2009-02-01 doi:10.1107/S0907444908020039 International Union of Crystallography In a first systematic exploration of phasing with Rosetta de novo models, it is shown that all-atom refinement of coarse-grained models significantly improves both the model quality and performance in molecular replacement with the Phaser software. en STRUCTURE PREDICTION; MOLECULAR REPLACEMENT; DE NOVO PHASING The prospect of phasing diffraction data sets `de novo' for proteins with previously unseen folds is appealing but largely untested. In a first systematic exploration of phasing with Rosetta de novo models, it is shown that all-atom refinement of coarse-grained models significantly improves both the model quality and performance in molecular replacement with the Phaser software. 15 new cases of diffraction data sets that are unambiguously phased with de novo models are presented. These diffraction data sets represent nine space groups and span a large range of solvent contents (33–79%) and asymmetric unit copy numbers (1–4). No correlation is observed between the ease of phasing and the solvent content or asymmetric unit copy number. Instead, a weak correlation is found with the length of the modeled protein: larger proteins required somewhat less accurate models to give successful molecular replacement. Overall, the results of this survey suggest that de novo models can phase diffraction data for approximately one sixth of proteins with sizes of 100 residues or less. However, for many of these cases, `de novo phasing with de novo models' requires significant investment of computational power, much greater than 103 CPU days per target. Improvements in conformational search methods will be necessary if molecular replacement with de novo models is to become a practical tool for targets without homology to previously solved protein structures. text/html Prospects for de novo phasing with de novo protein models text 65 2 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2009-02-01 169 research papers 0907-4449 med@iucr.org 175 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mv5020 A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies, subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Lawson, C.L. Dutta, S. Westbrook, J.D. Henrick, K. Berman, H.M. 2008-08-01 doi:10.1107/S0907444908017393 International Union of Crystallography A new data model for PDB entries of viruses and other biological assemblies with regular noncrystallographic symmetry is described. en VIRUS STRUCTURES; PROTEIN DATA BANK; DATABASE INTEGRATION; UNIFORM CURATION; POINT SYMMETRY; HELICAL SYMMETRY; BIOLOGICAL ASSEMBLIES A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies, subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive. text/html Representation of viruses in the remediated PDB archive text 64 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-08-01 874 research papers 0907-4449 med@iucr.org 882 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5129 The space-group symmetry of two crystal forms of rhodopsin (PDB codes 1gzm and 2j4y; space group P31) can be re-interpreted as hexagonal (space group P64). Two molecules of the G protein-coupled receptor are present in the asymmetric unit in the trigonal models. However, the noncrystallographic twofold axes parallel to the c axis can be treated as crystallographic symmetry operations in the hexagonal space group. This halves the asymmetric unit and makes all of the protein molecules equivalent in these structures. Corrections for merohedral twinning were also applied in the refinement in the higher symmetry space group for one of the structures (2j4y). http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Stenkamp, R.E. 2008-08-01 doi:10.1107/S0907444908017162 International Union of Crystallography Two crystal structures of rhodopsin that were originally described using trigonal symmetry can be interpreted in a hexagonal unit cell with a smaller asymmetric unit. en ALTERNATE SPACE GROUPS; RHODOPSIN; G PROTEIN-COUPLED RECEPTORS; INTEGRAL MEMBRANE PROTEINS The space-group symmetry of two crystal forms of rhodopsin (PDB codes 1gzm and 2j4y; space group P31) can be re-interpreted as hexagonal (space group P64). Two molecules of the G protein-coupled receptor are present in the asymmetric unit in the trigonal models. However, the noncrystallographic twofold axes parallel to the c axis can be treated as crystallographic symmetry operations in the hexagonal space group. This halves the asymmetric unit and makes all of the protein molecules equivalent in these structures. Corrections for merohedral twinning were also applied in the refinement in the higher symmetry space group for one of the structures (2j4y). text/html Alternative models for two crystal structures of bovine rhodopsin text 64 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-08-01 902 short communications 0907-4449 med@iucr.org 904 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx5126 An extremely low-field signal (at approximately 18 p.p.m.) in the 1H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theor­etical calculations of chemical shift values were used in the interpretation of the experimental 1H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor–acceptor distances of different types of hydrogen bonds. The short hydrogen-bond inter­actions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pKa values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Langkilde, A. Kristensen, S.M. Lo Leggio, L. Mølgaard, A. Jensen, J.H. Houk, A.R. Navarro Poulsen, J.-C. Kauppinen, S. Larsen, S. 2008-08-01 doi:10.1107/S0907444908017083 International Union of Crystallography The short hydrogen bonds in rhamnogalacturonan acetylesterase have been investigated by structure determination of an active-site mutant, 1H NMR spectra and computational methods. Comparisons are made to database statistics. A very short carboxylic acid carboxylate hydrogen bond, buried in the protein, could explain the low-field (18 p.p.m.) 1H NMR signal. en SHORT HYDROGEN BONDS; LOW-FIELD NMR SIGNALS; RHAMNOGALACTURONAN ACETYLESTERASE An extremely low-field signal (at approximately 18 p.p.m.) in the 1H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theor­etical calculations of chemical shift values were used in the interpretation of the experimental 1H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor–acceptor distances of different types of hydrogen bonds. The short hydrogen-bond inter­actions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pKa values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins. text/html Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase text 64 8 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-08-01 851 research papers 0907-4449 med@iucr.org 863 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5090 The X-ray polarization anisotropy of anomalous scattering in crystals of brominated nucleic acids and selenated proteins is shown to have significant effects on the diffraction data collected at an absorption edge. For conventionally collected single- or multi-wavelength anomalous diffraction data, the main manifestation of the anisotropy of anomalous scattering is the breakage of the equivalence between symmetry-related reflections, inducing intensity differences between them that can be exploited to yield extra phase information in the structure-solution process. A new formalism for describing the anisotropy of anomalous scattering which allows these effects to be incorporated into the general scheme of experimental phasing methods using an extended Harker construction is introduced. This requires a paradigm shift in the data-processing strategy, since the usual separation of the data-merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking is explicitly modelled and refined and becomes a source of supplementary phase information. These ideas have been implemented in the phasing program SHARP. Refinements using actual data show that exploitation of the anisotropy of anomalous scattering can deliver substantial extra phasing power compared with conventional approaches using the same raw data. Examples are given that show improvements in the phases which are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. It is argued that such gains, which come essentially for free, i.e. without the collection of new data, are highly significant, since radiation damage can frequently preclude the collection of a second-wavelength data set. Finally, further developments in synchrotron instrumentation and in the design of data-collection strategies that could help to maximize these gains are outlined. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Schiltz, M. Bricogne, G. 2008-07-01 doi:10.1107/S0907444908010202 International Union of Crystallography It is shown that the anisotropy of anomalous scattering (AAS) is a significant and ubiquitous effect in data sets collected at an absorption edge and that its exploitation can substantially enhance the phasing power of single- or multi-wavelength anomalous diffraction. The improvements in the phases are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. en ANISOTROPY OF ANOMALOUS SCATTERING; PHASING; SAD; MAD; POLARIZED RESONANT DIFFRACTION The X-ray polarization anisotropy of anomalous scattering in crystals of brominated nucleic acids and selenated proteins is shown to have significant effects on the diffraction data collected at an absorption edge. For conventionally collected single- or multi-wavelength anomalous diffraction data, the main manifestation of the anisotropy of anomalous scattering is the breakage of the equivalence between symmetry-related reflections, inducing intensity differences between them that can be exploited to yield extra phase information in the structure-solution process. A new formalism for describing the anisotropy of anomalous scattering which allows these effects to be incorporated into the general scheme of experimental phasing methods using an extended Harker construction is introduced. This requires a paradigm shift in the data-processing strategy, since the usual separation of the data-merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking is explicitly modelled and refined and becomes a source of supplementary phase information. These ideas have been implemented in the phasing program SHARP. Refinements using actual data show that exploitation of the anisotropy of anomalous scattering can deliver substantial extra phasing power compared with conventional approaches using the same raw data. Examples are given that show improvements in the phases which are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. It is argued that such gains, which come essentially for free, i.e. without the collection of new data, are highly significant, since radiation damage can frequently preclude the collection of a second-wavelength data set. Finally, further developments in synchrotron instrumentation and in the design of data-collection strategies that could help to maximize these gains are outlined. text/html Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments text 64 7 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-07-01 711 research papers 0907-4449 med@iucr.org 729 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5013 The first three-dimensional structure of a human Fc fragment genetically engineered for the elimination of its ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is reported. When introduced into the lower hinge and CH2 domain of human IgG1 molecules, the triple mutation L234F/L235E/P331S (`TM') causes a profound decrease in their binding to human CD64, CD32A, CD16 and C1q. Enzymatically produced Fc/TM fragment was crystallized and its structure was solved at a resolution of 2.3 Å using molecular replacement. This study revealed that the three-dimensional structure of Fc/TM is very similar to those of other human Fc fragments in the experimentally visible region spanning residues 236–­445. Thus, the dramatic broad-ranging effects of TM on IgG binding to several effector molecules cannot be explained in terms of major structural rearrangements in this portion of the Fc. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Oganesyan, V. Gao, C. Shirinian, L. Wu, H. Dall'Acqua, W.F. 2008-06-01 doi:10.1107/S0907444908007877 International Union of Crystallography Human Fc fragments containing the L234F/L235E/P331S triple mutation exhibit a dramatic decrease in their binding to several effector molecules (CD64, CD32A, CD16 and C1q). The three-dimensional structure of such a mutated fragment reveals that these broad-ranging functional effects are not caused by major structural rearrangements in the Fc moiety. en FC FRAGMENTS The first three-dimensional structure of a human Fc fragment genetically engineered for the elimination of its ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is reported. When introduced into the lower hinge and CH2 domain of human IgG1 molecules, the triple mutation L234F/L235E/P331S (`TM') causes a profound decrease in their binding to human CD64, CD32A, CD16 and C1q. Enzymatically produced Fc/TM fragment was crystallized and its structure was solved at a resolution of 2.3 Å using molecular replacement. This study revealed that the three-dimensional structure of Fc/TM is very similar to those of other human Fc fragments in the experimentally visible region spanning residues 236–­445. Thus, the dramatic broad-ranging effects of TM on IgG binding to several effector molecules cannot be explained in terms of major structural rearrangements in this portion of the Fc. text/html Structural characterization of a human Fc fragment engineered for lack of effector functions text 64 6 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-06-01 700 short communications 0907-4449 med@iucr.org 704 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5098 Interferon-γ-inducible protein (IP-10) belongs to the CXC class of chemokines and plays a significant role in the patho­physiology of various immune and inflammatory responses. It is also a potent angiostatic factor with antifibrotic properties. The biological activities of IP-10 are exerted by interactions with the G-protein-coupled receptor CXCR3 expressed on Th1 lymphocytes. IP-10 thus forms an attractive target for structure-based rational drug design of anti-inflammatory molecules. The crystal structure of mouse IP-­10 has been determined and reveals a novel tetrameric association. In the tetramer, two conventional CXC chemokine dimers are associated through their N-terminal regions to form a 12-­stranded elongated β-sheet of ∼90 Å in length. This association differs significantly from the previously studied tetramers of human IP-10, platelet factor 4 and neutrophil-activating peptide-2. In addition, heparin- and receptor-binding residues were mapped on the surface of IP-­10 tetramer. Two heparin-binding sites were observed on the surface and were present at the interface of each of the two β-­sheet dimers. The structure supports the formation of higher order oligomers of IP-10, as observed in recent in vivo studies with mouse IP-10, which will have functional relevance. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Jabeen, T. Leonard, P. Jamaluddin, H. Acharya, K.R. 2008-06-01 doi:10.1107/S0907444908007026 International Union of Crystallography The structure of mouse IP-10 shows a novel tetrameric association. en INTERFERON-[GAMMA]-INDUCIBLE PROTEIN; CHEMOKINES Interferon-γ-inducible protein (IP-10) belongs to the CXC class of chemokines and plays a significant role in the patho­physiology of various immune and inflammatory responses. It is also a potent angiostatic factor with antifibrotic properties. The biological activities of IP-10 are exerted by interactions with the G-protein-coupled receptor CXCR3 expressed on Th1 lymphocytes. IP-10 thus forms an attractive target for structure-based rational drug design of anti-inflammatory molecules. The crystal structure of mouse IP-­10 has been determined and reveals a novel tetrameric association. In the tetramer, two conventional CXC chemokine dimers are associated through their N-terminal regions to form a 12-­stranded elongated β-sheet of ∼90 Å in length. This association differs significantly from the previously studied tetramers of human IP-10, platelet factor 4 and neutrophil-activating peptide-2. In addition, heparin- and receptor-binding residues were mapped on the surface of IP-­10 tetramer. Two heparin-binding sites were observed on the surface and were present at the interface of each of the two β-­sheet dimers. The structure supports the formation of higher order oligomers of IP-10, as observed in recent in vivo studies with mouse IP-10, which will have functional relevance. text/html Structure of mouse IP-10, a chemokine text 64 6 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2008-06-01 611 research papers 0907-4449 med@iucr.org 619 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hm5059 The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented. The structure, solved at 2.0 Å resolution in complex with magnesium, reveals a well defined closed active site. Existing evidence suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for activation by Lys190 and attack on the substrate. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Parsons, J.F. Shi, K.M. Ladner, J.E. 2008-05-01 doi:10.1107/S0907444908005477 International Union of Crystallography The structure of the menaquinone-specific isochorismate synthase (MenF) from Escherichia coli has been refined at a resolution of 2.0 Å in complex with magnesium. The magnesium-bound structure has a well defined and organized active site which better represents the active conformation of the enzyme than the currently available structure. en CHORISMATE; ISOCHORISMATE; MENAQUINONE The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented. The structure, solved at 2.0 Å resolution in complex with magnesium, reveals a well defined closed active site. Existing evidence suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for activation by Lys190 and attack on the substrate. text/html Structure of isochorismate synthase in complex with magnesium text 64 5 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-05-01 607 short communications 0907-4449 med@iucr.org 610 1399-0047 http://scripts.iucr.org/cgi-bin/paper?mh5012 Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 Å, β = 124.8°. Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Kato, K. Tanaka, H. Sumizawa, T. Yoshimura, M. Yamashita, E. Iwasaki, K. Tsukihara, T. 2008-05-01 doi:10.1107/S0907444908004277 International Union of Crystallography A vault from rat liver was crystallized in space group C2. Rotational symmetry searches indicated that the particle has 39-fold dihedral symmetry. en VAULT; RIBONUCLEOPROTEINS Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 Å, β = 124.8°. Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry. text/html A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry text 64 5 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-05-01 525 research papers 0907-4449 med@iucr.org 531 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5088 A procedure for carrying out iterative model building, density modification and refinement is presented in which the density in an OMIT region is essentially unbiased by an atomic model. Density from a set of overlapping OMIT regions can be combined to create a composite `iterative-build' OMIT map that is everywhere unbiased by an atomic model but also everywhere benefiting from the model-based information present elsewhere in the unit cell. The procedure may have applications in the validation of specific features in atomic models as well as in overall model validation. The procedure is demonstrated with a molecular-replacement structure and with an experimentally phased structure and a variation on the method is demonstrated by removing model bias from a structure from the Protein Data Bank. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Terwilliger, T.C. Grosse-Kunstleve, R.W. Afonine, P.V. Moriarty, N.W. Adams, P.D. Read, R.J. Zwart, P.H. Hung, L.-W. 2008-05-01 doi:10.1107/S0907444908004319 International Union of Crystallography An OMIT procedure is presented that has the benefits of iterative model building density modification and refinement yet is essentially unbiased by the atomic model that is built. en MODEL BUILDING; MODEL VALIDATION; MACROMOLECULAR MODELS; PROTEIN DATA BANK; REFINEMENT; OMIT MAPS; BIAS; STRUCTURE REFINEMENT; PHENIX A procedure for carrying out iterative model building, density modification and refinement is presented in which the density in an OMIT region is essentially unbiased by an atomic model. Density from a set of overlapping OMIT regions can be combined to create a composite `iterative-build' OMIT map that is everywhere unbiased by an atomic model but also everywhere benefiting from the model-based information present elsewhere in the unit cell. The procedure may have applications in the validation of specific features in atomic models as well as in overall model validation. The procedure is demonstrated with a molecular-replacement structure and with an experimentally phased structure and a variation on the method is demonstrated by removing model bias from a structure from the Protein Data Bank. text/html Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias text 64 5 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-05-01 515 research papers 0907-4449 med@iucr.org 524 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hc5045 Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 Å structure of CYP46A1–CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 White, M.A. Mast, N. Bjorkhem, I. Johnson, E.F. Stout, C.D. Pikuleva, I.A. 2008-05-01 doi:10.1107/S0907444908004046 International Union of Crystallography Crystallization and analysis of the MIRAS heavy-atom structure solution of human cytochrome P450 46A1 using NaI and CsCl quick soaks. en CHOLESTEROL SULFATE; CHOLESTEROL HOMEOSTASIS; CYTOCHROME P450 46A1; MIRAS; MAD; MIR; HEAVY-ATOM DERIVATIVES; ALKALI-METAL SALTS; HALIDE SALTS Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 Å structure of CYP46A1–CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution. text/html Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1 text 64 5 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-05-01 487 research papers 0907-4449 med@iucr.org 495 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz0026 Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Karplus, P.A. Shapovalov, M.V. Dunbrack, R.L.Jr Berkholz, D.S. 2008-03-01 doi:10.1107/S0907444908002333 International Union of Crystallography A suggestion is put forward concerning the stereochemical restraints debate. en STEREOCHEMICAL RESTRAINTS; REFINEMENT text/html A forward-looking suggestion for resolving the stereochemical restraints debate: ideal geometry functions text 64 3 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-03-01 335 letters to the editor 0907-4449 med@iucr.org 336 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx5125 One of the most cumbersome and time-demanding tasks in completing a protein model is building short missing regions or `loops'. A method is presented that uses structural and electron-density information to build the most likely conformations of such loops. Using the distribution of angles and dihedral angles in pentapeptides as the driving parameters, a set of possible conformations for the Cα backbone of loops was generated. The most likely candidate is then selected in a hierarchical manner: new and stronger restraints are added while the loop is built. The weight of the electron-density correlation relative to geometrical considerations is gradually increased until the most likely loop is selected on map correlation alone. To conclude, the loop is refined against the electron density in real space. This is started by using structural information to trace a set of models for the Cα backbone of the loop. Only in later steps of the algorithm is the electron-density correlation used as a criterion to select the loop(s). Thus, this method is more robust in low-density regions than an approach using density as a primary criterion. The algorithm is implemented in a loop-building program, Loopy, which can be used either alone or as part of an automatic building cycle. Loopy can build loops of up to 14 residues in length within a couple of minutes. The average root-mean-square deviation of the Cα atoms in the loops built during validation was less than 0.4 Å. When implemented in the context of automated model building in ARP/wARP, Loopy can increase the completeness of the built models. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Joosten, K. Cohen, S.X. Emsley, P. Mooij, W. Lamzin, V.S. Perrakis, A. 2008-04-01 doi:10.1107/S0907444908001558 International Union of Crystallography A novel method that uses the conformational distribution of Cα atoms in known structures is used to build short missing regions (`loops') in protein models. An initial tree of possible loop paths is pruned according to structural and electron-density criteria and the most likely loop conformation(s) are selected and built. en MODEL BUILDING; LOOP MODELLING; LOOPY One of the most cumbersome and time-demanding tasks in completing a protein model is building short missing regions or `loops'. A method is presented that uses structural and electron-density information to build the most likely conformations of such loops. Using the distribution of angles and dihedral angles in pentapeptides as the driving parameters, a set of possible conformations for the Cα backbone of loops was generated. The most likely candidate is then selected in a hierarchical manner: new and stronger restraints are added while the loop is built. The weight of the electron-density correlation relative to geometrical considerations is gradually increased until the most likely loop is selected on map correlation alone. To conclude, the loop is refined against the electron density in real space. This is started by using structural information to trace a set of models for the Cα backbone of the loop. Only in later steps of the algorithm is the electron-density correlation used as a criterion to select the loop(s). Thus, this method is more robust in low-density regions than an approach using density as a primary criterion. The algorithm is implemented in a loop-building program, Loopy, which can be used either alone or as part of an automatic building cycle. Loopy can build loops of up to 14 residues in length within a couple of minutes. The average root-mean-square deviation of the Cα atoms in the loops built during validation was less than 0.4 Å. When implemented in the context of automated model building in ARP/wARP, Loopy can increase the completeness of the built models. text/html A knowledge-driven approach for crystallographic protein model completion text 64 4 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-04-01 416 research papers 0907-4449 med@iucr.org 424 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hm5052 Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Soriano, E.V. McCloskey, D.E. Kinsland, C. Pegg, A.E. Ealick, S.E. 2008-04-01 doi:10.1107/S0907444908000474 International Union of Crystallography The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. en ARGININE DECARBOXYLASE; PYRUVOYL; DECARBOXYLATION; AUTOPROCESSING; SERINOLYSIS Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction. text/html Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii text 64 4 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-04-01 377 research papers 0907-4449 med@iucr.org 382 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5082 For the first time, protein microcrystallography has been performed with a focused synchrotron-radiation beam of 1 µm using a goniometer with a sub-micrometre sphere of confusion. The crystal structure of xylanase II has been determined with a flux density of about 3 × 1010 photons s−1 µm−2 at the sample. Two sets of diffraction images collected from different sized crystals were shown to comprise data of good quality, which allowed a 1.5 Å resolution xylanase II structure to be obtained. The main conclusion of this experiment is that a high-resolution diffraction pattern can be obtained from 20 µm3 crystal volume, corresponding to about 2 × 108 unit cells. Despite the high irradiation dose in this case, it was possible to obtain an excellent high-resolution map and it could be concluded from the individual atomic B-factor patterns that there was no evidence of significant radiation damage. The photoelectron escape from a narrow diffraction channel is a possible reason for reduced radiation damage as indicated by Monte Carlo simulations. These results open many new opportunities in scanning protein microcrystallography and make random data collection from microcrystals a real possibility, therefore enabling structures to be solved from much smaller crystals than previously anticipated as long as the crystallites are well ordered. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Moukhametzianov, R. Burghammer, M. Edwards, P.C. Petitdemange, S. Popov, D. Fransen, M. McMullan, G. Schertler, G.F.X. Riekel, C. 2008-02-01 doi:10.1107/S090744490705812X International Union of Crystallography For the first time, protein microcrystallography has been performed with a focused synchrotron-radiation beam of 1 µm using a goniometer with a sub-micrometre sphere of confusion. The crystal structure of xylanase II has been determined with a flux density of about 3 × 1010 photons s−1 µm−2 at the sample. en MICROCRYSTALLOGRAPHY; XYLANASE II For the first time, protein microcrystallography has been performed with a focused synchrotron-radiation beam of 1 µm using a goniometer with a sub-micrometre sphere of confusion. The crystal structure of xylanase II has been determined with a flux density of about 3 × 1010 photons s−1 µm−2 at the sample. Two sets of diffraction images collected from different sized crystals were shown to comprise data of good quality, which allowed a 1.5 Å resolution xylanase II structure to be obtained. The main conclusion of this experiment is that a high-resolution diffraction pattern can be obtained from 20 µm3 crystal volume, corresponding to about 2 × 108 unit cells. Despite the high irradiation dose in this case, it was possible to obtain an excellent high-resolution map and it could be concluded from the individual atomic B-factor patterns that there was no evidence of significant radiation damage. The photoelectron escape from a narrow diffraction channel is a possible reason for reduced radiation damage as indicated by Monte Carlo simulations. These results open many new opportunities in scanning protein microcrystallography and make random data collection from microcrystals a real possibility, therefore enabling structures to be solved from much smaller crystals than previously anticipated as long as the crystallites are well ordered. text/html Protein crystallography with a micrometre-sized synchrotron-radiation beam text 64 2 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-02-01 158 research papers 0907-4449 med@iucr.org 166 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5111 It is not uncommon for protein crystals to crystallize with more than a single molecule per asymmetric unit. When more than a single molecule is present in the asymmetric unit, various pathological situations such as twinning, modulated crystals and pseudo translational or rotational symmetry can arise. The presence of pseudosymmetry can lead to uncertainties about the correct space group, especially in the presence of twinning. The background to certain common pathologies is presented and a new notation for space groups in unusual settings is introduced. The main concepts are illustrated with several examples from the literature and the Protein Data Bank. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Zwart, P.H. Grosse-Kunstleve, R.W. Lebedev, A.A. Murshudov, G.N. Adams, P.D. 2008-01-01 doi:10.1107/S090744490705531X International Union of Crystallography The presence of pseudosymmetry can cause problems in structure determination and refinement. The relevant background and representative examples are presented. en PATHOLOGY; TWINNING; PSEUDOSYMMETRY It is not uncommon for protein crystals to crystallize with more than a single molecule per asymmetric unit. When more than a single molecule is present in the asymmetric unit, various pathological situations such as twinning, modulated crystals and pseudo translational or rotational symmetry can arise. The presence of pseudosymmetry can lead to uncertainties about the correct space group, especially in the presence of twinning. The background to certain common pathologies is presented and a new notation for space groups in unusual settings is introduced. The main concepts are illustrated with several examples from the literature and the Protein Data Bank. text/html Surprises and pitfalls arising from (pseudo)symmetry text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 99 research papers 0907-4449 med@iucr.org 107 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5112 Type II dehydroquinase is a small (150-amino-acid) protein which in solution packs together to form a dodecamer with 23 cubic symmetry. In crystals of this protein the symmetry of the biological unit can be coincident with the crystallographic symmetry, giving rise to cubic crystal forms with a single monomer in the asymmetric unit. In crystals where this is not the case, multiple copies of the monomer are present, giving rise to significant and often confusing noncrystallographic symmetry in low-symmetry crystal systems. These different crystal forms pose a variety of challenges for solution by molecular replacement. Three examples of structure solutions, including a highly unusual triclinic crystal form with 16 dodecamers (192 monomers) in the unit cell, are described. Four commonly used molecular-replacement packages are assessed against two of these examples, one of high symmetry and the other of low symmetry; this study highlights how program performance can vary significantly depending on the given problem. In addition, the final refined structure of the 16-dodecamer triclinic crystal form is analysed and shown not to be a superlattice structure, but rather an F-centred cubic crystal with frustrated crystallographic symmetry. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Stewart, K.A. Robinson, D.A. Lapthorn, A.J. 2008-01-01 doi:10.1107/S0907444907054923 International Union of Crystallography The type II dehydroquinase enzyme is a symmetrical dodecameric protein which crystallizes in either high-symmetry cubic space groups or low-symmetry crystal systems with multiple copies in the asymmetric unit. Both systems have provided challenging examples for molecular replacement; for example, a triclinic crystal form has 16 dodecamers (192 monomers) in the unit cell. Three difficult examples are discussed and two are used as test cases to compare the performance of four commonly used molecular-replacement packages. en MULTI-COPY MOLECULAR REPLACEMENT; SUPERLATTICE STRUCTURE; PSEUDO-CUBIC SYMMETRY; TYPE II DEHYDROQUINASES Type II dehydroquinase is a small (150-amino-acid) protein which in solution packs together to form a dodecamer with 23 cubic symmetry. In crystals of this protein the symmetry of the biological unit can be coincident with the crystallographic symmetry, giving rise to cubic crystal forms with a single monomer in the asymmetric unit. In crystals where this is not the case, multiple copies of the monomer are present, giving rise to significant and often confusing noncrystallographic symmetry in low-symmetry crystal systems. These different crystal forms pose a variety of challenges for solution by molecular replacement. Three examples of structure solutions, including a highly unusual triclinic crystal form with 16 dodecamers (192 monomers) in the unit cell, are described. Four commonly used molecular-replacement packages are assessed against two of these examples, one of high symmetry and the other of low symmetry; this study highlights how program performance can vary significantly depending on the given problem. In addition, the final refined structure of the 16-dodecamer triclinic crystal form is analysed and shown not to be a superlattice structure, but rather an F-centred cubic crystal with frustrated crystallographic symmetry. text/html Type II dehydroquinase: molecular replacement with many copies text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 108 research papers 0907-4449 med@iucr.org 118 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5106 Multi-component molecular complexes are increasingly being tackled by structural biology, bringing X-ray crystallography into the purview of electron-microscopy (EM) studies. X-ray crystallography can utilize a low-resolution EM map for structure determination followed by phase extension to high resolution. Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement (MR) using various standard MR packages such as AMoRe, MOLREP and Phaser. The results demonstrate that EM maps are viable models for molecular replacement. Possible difficulties in data analysis, such as the effects of the EM magnification error, and the effect of MR positional/rotational errors on phase extension are discussed. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Xiong, Y. 2008-01-01 doi:10.1107/S090744490705398X International Union of Crystallography Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement using various standard MR packages such as AMoRe, MOLREP and Phaser. en ELECTRON MICROSCOPY; MOLECULAR REPLACEMENT Multi-component molecular complexes are increasingly being tackled by structural biology, bringing X-ray crystallography into the purview of electron-microscopy (EM) studies. X-ray crystallography can utilize a low-resolution EM map for structure determination followed by phase extension to high resolution. Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement (MR) using various standard MR packages such as AMoRe, MOLREP and Phaser. The results demonstrate that EM maps are viable models for molecular replacement. Possible difficulties in data analysis, such as the effects of the EM magnification error, and the effect of MR positional/rotational errors on phase extension are discussed. text/html From electron microscopy to X-ray crystallography: molecular-replacement case studies text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 76 research papers 0907-4449 med@iucr.org 82 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5120 The efficiency of the cross-rotation function step of molecular replacement (MR) is intrinsically limited as it uses only a fraction of the Patterson vectors. Along with general techniques extending the boundaries of the method, there are approaches that utilize specific features of a given structure. In special cases, where the directions of noncrystallographic symmetry axes can be unambiguously derived from the self-rotation function and the structure of the homologue protein is available in a related oligomeric state, the cross-rotation function step of MR can be omitted. In such cases, a small number of yet unknown parameters defining the orientation of the oligomer and/or its internal organization can be optimized using an exhaustive search. Three difficult MR cases are reported in which these parameters were determined and the oligomer was positioned according to the maximal value of the correlation coefficient in a series of translation searches. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Isupov, M.N. Lebedev, A.A. 2008-01-01 doi:10.1107/S0907444907053802 International Union of Crystallography Three difficult MR cases are reported in which the orientation of a search oligomer or its internal parameters were determined and the oligomer was positioned according to the maximal value of the correlation coefficient in a series of translation searches. Such an exhaustive search was feasible because of constraints on the model parameters derived from the self-rotation function. en MOLECULAR REPLACEMENT; EXHAUSTIVE SEARCH The efficiency of the cross-rotation function step of molecular replacement (MR) is intrinsically limited as it uses only a fraction of the Patterson vectors. Along with general techniques extending the boundaries of the method, there are approaches that utilize specific features of a given structure. In special cases, where the directions of noncrystallographic symmetry axes can be unambiguously derived from the self-rotation function and the structure of the homologue protein is available in a related oligomeric state, the cross-rotation function step of MR can be omitted. In such cases, a small number of yet unknown parameters defining the orientation of the oligomer and/or its internal organization can be optimized using an exhaustive search. Three difficult MR cases are reported in which these parameters were determined and the oligomer was positioned according to the maximal value of the correlation coefficient in a series of translation searches. text/html NCS-constrained exhaustive search using oligomeric models text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 90 research papers 0907-4449 med@iucr.org 98 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5110 Normal-mode analysis (NMA) can be used to generate multiple structural variants of a given template model, thereby increasing the chance of finding the molecular-replacement solution. Here, it is shown that it is also possible to directly refine the amplitudes of the normal modes against experimental data (X-ray or cryo-EM), generalizing rigid-body refinement methods by adding just a few additional degrees of freedom that sample collective and large-amplitude movements. It is also argued that the situation where several (conformations of) models are present simultaneously in the crystal can be studied with adjustable occupancies using techniques derived from statistical thermodynamics and already used in molecular modelling. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Delarue, M. 2008-01-01 doi:10.1107/S0907444907053516 International Union of Crystallography The possibility of taking into account large-amplitude collective movements of a given model by using a subset of low-frequency normal modes is evaluated for molecular replacement and refinement using X-ray data or cryo-EM maps. en NORMAL-MODE ANALYSIS; MOLECULAR REPLACEMENT; REFINEMENT Normal-mode analysis (NMA) can be used to generate multiple structural variants of a given template model, thereby increasing the chance of finding the molecular-replacement solution. Here, it is shown that it is also possible to directly refine the amplitudes of the normal modes against experimental data (X-ray or cryo-EM), generalizing rigid-body refinement methods by adding just a few additional degrees of freedom that sample collective and large-amplitude movements. It is also argued that the situation where several (conformations of) models are present simultaneously in the crystal can be studied with adjustable occupancies using techniques derived from statistical thermodynamics and already used in molecular modelling. text/html Dealing with structural variability in molecular replacement and crystallographic refinement through normal-mode analysis text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 40 research papers 0907-4449 med@iucr.org 48 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5118 Low-resolution electron-microscopy reconstructions can be used as search models in molecular replacement or may be combined with existing monomeric structures in order to produce multimeric models suitable for molecular replacement. The technique is described in the case of viral and subviral particles as well as in the case of oligomeric proteins. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Navaza, J. 2008-01-01 doi:10.1107/S0907444907053334 International Union of Crystallography Overview and examples of combined use of X-ray and electron-microscopy data. en ELECTRON MICROSCOPY; MOLECULAR REPLACEMENT Low-resolution electron-microscopy reconstructions can be used as search models in molecular replacement or may be combined with existing monomeric structures in order to produce multimeric models suitable for molecular replacement. The technique is described in the case of viral and subviral particles as well as in the case of oligomeric proteins. text/html Combining X-ray and electron-microscopy data to solve crystal structures text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 70 research papers 0907-4449 med@iucr.org 75 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5108 Molecular replacement is fundamentally a simple trial-and-error method of solving crystal structures when a suitable related model is available. The underlying simplicity of the method is often obscured by the mathematical trickery required to make the searches computationally tractable. This introduction sketches the essential issues in molecular replacement without going into technical details. General search strategies are discussed and the alternative Patterson and likelihood approaches are outlined. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Evans, P. McCoy, A. 2008-01-01 doi:10.1107/S0907444907051554 International Union of Crystallography An outline is given of the basic features of the molecular-replacement method for solving crystal structures. en MOLECULAR REPLACEMENT; ROTATION FUNCTION; TRANSLATION FUNCTION; MAXIMUM LIKELIHOOD Molecular replacement is fundamentally a simple trial-and-error method of solving crystal structures when a suitable related model is available. The underlying simplicity of the method is often obscured by the mathematical trickery required to make the searches computationally tractable. This introduction sketches the essential issues in molecular replacement without going into technical details. General search strategies are discussed and the alternative Patterson and likelihood approaches are outlined. text/html An introduction to molecular replacement text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 1 research papers 0907-4449 med@iucr.org 10 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5109 The PHENIX AutoBuild wizard is a highly automated tool for iterative model building, structure refinement and density modification using RESOLVE model building, RESOLVE statistical density modification and phenix.refine structure refinement. Recent advances in the AutoBuild wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model-completion algorithms and automated solvent-molecule picking. Model-completion algorithms in the AutoBuild wizard include loop building, crossovers between chains in different models of a structure and side-chain optimization. The AutoBuild wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 to 3.2 Å, resulting in a mean R factor of 0.24 and a mean free R factor of 0.29. The R factor of the final model is dependent on the quality of the starting electron density and is relatively independent of resolution. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Terwilliger, T.C. Grosse-Kunstleve, R.W. Afonine, P.V. Moriarty, N.W. Zwart, P.H. Hung, L.-W. Read, R.J. Adams, P.D. 2008-01-01 doi:10.1107/S090744490705024X International Union of Crystallography The highly automated PHENIX AutoBuild wizard is described. The procedure can be applied equally well to phases derived from isomorphous/anomalous and molecular-replacement methods. en MODEL BUILDING; MODEL COMPLETION; MACROMOLECULAR MODELS; PROTEIN DATA BANK; STRUCTURE REFINEMENT; PHENIX The PHENIX AutoBuild wizard is a highly automated tool for iterative model building, structure refinement and density modification using RESOLVE model building, RESOLVE statistical density modification and phenix.refine structure refinement. Recent advances in the AutoBuild wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model-completion algorithms and automated solvent-molecule picking. Model-completion algorithms in the AutoBuild wizard include loop building, crossovers between chains in different models of a structure and side-chain optimization. The AutoBuild wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 to 3.2 Å, resulting in a mean R factor of 0.24 and a mean free R factor of 0.29. The R factor of the final model is dependent on the quality of the starting electron density and is relatively independent of resolution. text/html Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 61 research papers 0907-4449 med@iucr.org 69 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx5119 A number of inconsistencies are apparent in the recent research paper by Jaskolski et al. [(2007), Acta Cryst. D63, 611–620] concerning their recommendations for the values of the magnitude and resolution-dependence of the root-mean-square deviations (RMSDs) of bond lengths and angles from their restrained ideal values in macromolecular refinement, as well as their suggestions for the use of variable standard uncertainties dependent on atomic displacement parameters (ADPs) and occupancies. Whilst many of the comments and suggestions in the paper regarding updates for the ideal geometry values proposed by Engh and Huber are entirely reasonable and supported by the experimental evidence, the recommendations concerning the optimal values of RMSDs appear to be in conflict with previous experimental and theoretical work in this area [Tickle et al. (1998), Acta Cryst. D54, 243–252] and indeed appear to be based on a misunderstanding of the distinction between RMSD and standard uncertainty (SU). In contrast, it is proposed here that the optimal values of all desired weighting parameters, in particular the weighting parameters for the ADP differences and for the diffraction terms, be estimated by the purely objective procedure of maximizing the experiment-based log(free likelihood). In principle, this allows all weighting parameters that are not known accurately a priori to be scaled globally, relative to those that are known accurately, for an optimal refinement. The RMS Z score (RMSZ) is recommended as a more satisfactory statistic than the RMSD to assess the extent to which the geometry deviates from the ideal values and a theoretical rationale for the results obtained is presented in which the optimal RMSZ is identified as the calculated versus true Z-score correlation coefficient, the latter being a monotonic function of the resolution cutoff of the data. Regarding the proposal to use variable standard uncertainties, it is suggested that any departure from the current practice of using fixed weights for geometric restraints based on experimental values of standard uncertainties be subject to the same experiment-based validation. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Tickle, I.J. 2007-12-01 doi:10.1107/S0907444907050196 International Union of Crystallography Comment on Jaskolski et al. [(2007), Acta Cryst. D63, 611–620]. en MACROMOLECULAR REFINEMENT; GEOMETRIC RESTRAINTS; ROOT-MEAN-SQUARE DEVIATION; OPTIMAL WEIGHTING PARAMETERS; CROSS-VALIDATION; FREE LIKELIHOOD A number of inconsistencies are apparent in the recent research paper by Jaskolski et al. [(2007), Acta Cryst. D63, 611–620] concerning their recommendations for the values of the magnitude and resolution-dependence of the root-mean-square deviations (RMSDs) of bond lengths and angles from their restrained ideal values in macromolecular refinement, as well as their suggestions for the use of variable standard uncertainties dependent on atomic displacement parameters (ADPs) and occupancies. Whilst many of the comments and suggestions in the paper regarding updates for the ideal geometry values proposed by Engh and Huber are entirely reasonable and supported by the experimental evidence, the recommendations concerning the optimal values of RMSDs appear to be in conflict with previous experimental and theoretical work in this area [Tickle et al. (1998), Acta Cryst. D54, 243–252] and indeed appear to be based on a misunderstanding of the distinction between RMSD and standard uncertainty (SU). In contrast, it is proposed here that the optimal values of all desired weighting parameters, in particular the weighting parameters for the ADP differences and for the diffraction terms, be estimated by the purely objective procedure of maximizing the experiment-based log(free likelihood). In principle, this allows all weighting parameters that are not known accurately a priori to be scaled globally, relative to those that are known accurately, for an optimal refinement. The RMS Z score (RMSZ) is recommended as a more satisfactory statistic than the RMSD to assess the extent to which the geometry deviates from the ideal values and a theoretical rationale for the results obtained is presented in which the optimal RMSZ is identified as the calculated versus true Z-score correlation coefficient, the latter being a monotonic function of the resolution cutoff of the data. Regarding the proposal to use variable standard uncertainties, it is suggested that any departure from the current practice of using fixed weights for geometric restraints based on experimental values of standard uncertainties be subject to the same experiment-based validation. text/html Experimental determination of optimal root-mean-square deviations of macromolecular bond lengths and angles from their restrained ideal values text 63 12 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-12-01 1274 letters to the editor 0907-4449 med@iucr.org 1281 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5107 The success rate of molecular replacement (MR) falls considerably when search models share less than 35% sequence identity with their templates, but can be improved significantly by using fold-recognition methods combined with exhaustive MR searches. Models based on alignments calculated with fold-recognition algorithms are more accurate than models based on conventional alignment methods such as FASTA or BLAST, which are still widely used for MR. In addition, by designing MR pipelines that integrate phasing and automated refinement and allow parallel processing of such calculations, one can effectively increase the success rate of MR. Here, updated results from the JCSG MR pipeline are presented, which to date has solved 33 MR structures with less than 35% sequence identity to the closest homologue of known structure. By using difficult MR problems as examples, it is demonstrated that successful MR phasing is possible even in cases where the similarity between the model and the template can only be detected with fold-recognition algorithms. In the first step, several search models are built based on all homologues found in the PDB by fold-recognition algorithms. The models resulting from this process are used in parallel MR searches with different combinations of input parameters of the MR phasing algorithm. The putative solutions are subjected to rigid-body and restrained crystallo­graphic refinement and ranked based on the final values of free R factor, figure of merit and deviations from ideal geometry. Finally, crystal packing and electron-density maps are checked to identify the correct solution. If this procedure does not yield a solution with interpretable electron-density maps, then even more alternative models are prepared. The structurally variable regions of a protein family are identified based on alignments of sequences and known structures from that family and appropriate trimmings of the models are proposed. All combinations of these trimmings are applied to the search models and the resulting set of models is used in the MR pipeline. It is estimated that with the improvements in model building and exhaustive parallel searches with existing phasing algorithms, MR can be successful for more than 50% of recognizable homologues of known structures below the threshold of 35% sequence identity. This implies that about one-third of the proteins in a typical bacterial proteome are potential MR targets. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Schwarzenbacher, R. Godzik, A. Jaroszewski, L. 2008-01-01 doi:10.1107/S0907444907050111 International Union of Crystallography The practical limits of molecular replacement can be extended by using several specifically designed protein models based on fold-recognition methods and by exhaustive searches performed in a parallelized pipeline. Updated results from the JCSG MR pipeline, which to date has solved 33 molecular-replacement structures with less than 35% sequence identity to the closest homologue of known structure, are presented. en MOLECULAR REPLACEMENT; SEQUENCE-ALIGNMENT ACCURACY; HOMOLOGY MODELING; PARAMETER-SPACE SCREENING; STRUCTURAL GENOMICS The success rate of molecular replacement (MR) falls considerably when search models share less than 35% sequence identity with their templates, but can be improved significantly by using fold-recognition methods combined with exhaustive MR searches. Models based on alignments calculated with fold-recognition algorithms are more accurate than models based on conventional alignment methods such as FASTA or BLAST, which are still widely used for MR. In addition, by designing MR pipelines that integrate phasing and automated refinement and allow parallel processing of such calculations, one can effectively increase the success rate of MR. Here, updated results from the JCSG MR pipeline are presented, which to date has solved 33 MR structures with less than 35% sequence identity to the closest homologue of known structure. By using difficult MR problems as examples, it is demonstrated that successful MR phasing is possible even in cases where the similarity between the model and the template can only be detected with fold-recognition algorithms. In the first step, several search models are built based on all homologues found in the PDB by fold-recognition algorithms. The models resulting from this process are used in parallel MR searches with different combinations of input parameters of the MR phasing algorithm. The putative solutions are subjected to rigid-body and restrained crystallo­graphic refinement and ranked based on the final values of free R factor, figure of merit and deviations from ideal geometry. Finally, crystal packing and electron-density maps are checked to identify the correct solution. If this procedure does not yield a solution with interpretable electron-density maps, then even more alternative models are prepared. The structurally variable regions of a protein family are identified based on alignments of sequences and known structures from that family and appropriate trimmings of the models are proposed. All combinations of these trimmings are applied to the search models and the resulting set of models is used in the MR pipeline. It is estimated that with the improvements in model building and exhaustive parallel searches with existing phasing algorithms, MR can be successful for more than 50% of recognizable homologues of known structures below the threshold of 35% sequence identity. This implies that about one-third of the proteins in a typical bacterial proteome are potential MR targets. text/html The JCSG MR pipeline: optimized alignments, multiple models and parallel searches text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 133 research papers 0907-4449 med@iucr.org 140 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5114 The number of macromolecular structures solved and deposited in the Protein Data Bank (PDB) is higher than 40 000. Using this information in macromolecular crystallo­graphy (MX) should in principle increase the efficiency of MX structure solution. This paper describes a molecular-replacement pipeline, BALBES, that makes extensive use of this repository. It uses a reorganized database taken from the PDB with multimeric as well as domain organization. A system manager written in Python controls the workflow of the process. Testing the current version of the pipeline using entries from the PDB has shown that this approach has huge potential and that around 75% of structures can be solved automatically without user intervention. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Long, F. Vagin, A.A. Young, P. Murshudov, G.N. 2008-01-01 doi:10.1107/S0907444907050172 International Union of Crystallography The fully automated pipeline, BALBES, integrates a redesigned hierarchical database of protein structures with their domains and multimeric organization, and solves molecular-replacement problems using only input X-ray and sequence data. en BALBES; MOLECULAR REPLACEMENT The number of macromolecular structures solved and deposited in the Protein Data Bank (PDB) is higher than 40 000. Using this information in macromolecular crystallo­graphy (MX) should in principle increase the efficiency of MX structure solution. This paper describes a molecular-replacement pipeline, BALBES, that makes extensive use of this repository. It uses a reorganized database taken from the PDB with multimeric as well as domain organization. A system manager written in Python controls the workflow of the process. Testing the current version of the pipeline using entries from the PDB has shown that this approach has huge potential and that around 75% of structures can be solved automatically without user intervention. text/html BALBES: a molecular-replacement pipeline text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 125 research papers 0907-4449 med@iucr.org 132 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5117 The success of molecular replacement is critically dependent on the quality of the search model. Several model-preparation procedures are integrated in the molecular-replacement program MOLREP. These include model modification on the basis of amino-acid sequence alignment and model correction based on analysis of the solvent-accessibility of the atoms. The packing function used in MOLREP for the translational search is explained in the context of model preparation. In difficult cases, bioinformatics-based modifications are not sufficient for successful molecular replacement. An approach implemented in MOLREP for solving cases with translational noncrystallographic symmetry is an example of model preparation in which analysis of X-ray data plays an essential role. In addition, two examples are presented in which the X-ray data were used to refine partial models for subsequent use in molecular replacement. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Lebedev, A.A. Vagin, A.A. Murshudov, G.N. 2008-01-01 doi:10.1107/S0907444907049839 International Union of Crystallography The default model-preparation scheme of MOLREP is described. Two examples are presented of model improvement using X-ray data. en MOLREP; MODEL PREPARATION; MOLECULAR REPLACEMENT The success of molecular replacement is critically dependent on the quality of the search model. Several model-preparation procedures are integrated in the molecular-replacement program MOLREP. These include model modification on the basis of amino-acid sequence alignment and model correction based on analysis of the solvent-accessibility of the atoms. The packing function used in MOLREP for the translational search is explained in the context of model preparation. In difficult cases, bioinformatics-based modifications are not sufficient for successful molecular replacement. An approach implemented in MOLREP for solving cases with translational noncrystallographic symmetry is an example of model preparation in which analysis of X-ray data plays an essential role. In addition, two examples are presented in which the X-ray data were used to refine partial models for subsequent use in molecular replacement. text/html Model preparation in MOLREP and examples of model improvement using X-ray data text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 33 research papers 0907-4449 med@iucr.org 39 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5090 Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by β-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25 µM as determined by fluorescence titration. A 1.35 Å crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein–ligand interaction offers strategies for further optimization of this low-molecular-weight compound. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Pappenberger, G. Schulz-Gasch, T. Kusznir, E. Müller, F. Hennig, M. 2007-12-01 doi:10.1107/S0907444907049852 International Union of Crystallography β-Ketoacyl-ACP synthase is a key target for the treatment of infectious diseases. A structure-based biophysical screening approach identified for the first time a synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to the active site of the enzyme. Implications for the use of this information in drug discovery are discussed. en [BETA]-KETOACYL-ACP SYNTHASE; DRUG DESIGN; FATTY-ACID SYNTHESIS; ANTIBIOTICS Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by β-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25 µM as determined by fluorescence titration. A 1.35 Å crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein–ligand interaction offers strategies for further optimization of this low-molecular-weight compound. text/html Structure-assisted discovery of an aminothiazole derivative as a lead molecule for inhibition of bacterial fatty-acid synthesis text 63 12 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-12-01 1208 research papers 0907-4449 med@iucr.org 1216 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5119 This review addresses the essential questions to consider when attempting to phase a new crystal structure using molecular replacement. Sequence matching can suggest whether there is a suitable three-dimensional model available, but it is also important to analyse the model in order to find its likely oligomeric state and to establish whether there are likely to be domain movements. Once a solution has been found it must be refined, which can be challenging for low-homology models. There is a detailed discussion of structures used as examples for CCP4 tutorials. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Dodson, E. 2008-01-01 doi:10.1107/S0907444907049736 International Union of Crystallography This review outlines questions to consider when attempting to solve crystal structures by molecular replacement. en BIOINFORMATICS; MOLECULAR REPLACEMENT; VALIDATION This review addresses the essential questions to consider when attempting to phase a new crystal structure using molecular replacement. Sequence matching can suggest whether there is a suitable three-dimensional model available, but it is also important to analyse the model in order to find its likely oligomeric state and to establish whether there are likely to be domain movements. Once a solution has been found it must be refined, which can be challenging for low-homology models. There is a detailed discussion of structures used as examples for CCP4 tutorials. text/html The befores and afters of molecular replacement text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 17 research papers 0907-4449 med@iucr.org 24 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5098 Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Jaskolski, M. Gilski, M. Dauter, Z. Wlodawer, A. 2007-12-01 doi:10.1107/S0907444907049359 International Union of Crystallography Response to comments by Stec [(2007), Acta Cryst. D63, 1113–1114] and Tickle [(2007), Acta Cryst. D63, 1274–1281] on Jaskolski et al. [(2007), Acta Cryst. D63, 611–620]. en STEREOCHEMICAL RESTRAINTS; REFINEMENT text/html Numerology versus reality: a voice in a recent dispute text 63 12 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-12-01 1282 letters to the editor 0907-4449 med@iucr.org 1283 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5116 Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Cohen, S.X. Ben Jelloul, M. Long, F. Vagin, A. Knipscheer, P. Lebbink, J. Sixma, T.K. Lamzin, V.S. Murshudov, G.N. Perrakis, A. 2008-01-01 doi:10.1107/S0907444907047580 International Union of Crystallography A systematic test shows how ARP/wARP deals with automated model building for structures that have been solved by molecular replacement. A description of protocols in the flex-wARP control system and studies of two specific cases are also presented. en MODEL BUILDING; REFINEMENT; MOLECULAR REPLACEMENT Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information. text/html ARP/wARP and molecular replacement: the next generation text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 49 research papers 0907-4449 med@iucr.org 60 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz5115 A study of the accurate electron-density distribution in molecular crystals at subatomic resolution (better than ∼1.0 Å) requires more detailed models than those based on independent spherical atoms. A tool that is conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8–1.0 Å, the number of experimental data is insufficient for full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark data sets gave results that were comparable in quality with the results of multipolar refinement and superior to those for conventional models. Applications to several data sets of both small molecules and macromolecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package. http://creativecommons.org/licenses/by/2.0/uk urn:issn:0907-4449 Afonine, P.V. Grosse-Kunstleve, R.W. Adams, P.D. Lunin, V.Y. Urzhumtsev, A. 2007-11-01 doi:10.1107/S0907444907046148 International Union of Crystallography Modelling deformation electron density using interatomic scatters is simpler than multipolar methods, produces comparable results at subatomic resolution and can easily be applied to macromolecules. en STRUCTURE REFINEMENT; SUBATOMIC RESOLUTION; DEFORMATION DENSITY; INTERATOMIC SCATTERERS; PHENIX A study of the accurate electron-density distribution in molecular crystals at subatomic resolution (better than ∼1.0 Å) requires more detailed models than those based on independent spherical atoms. A tool that is conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8–1.0 Å, the number of experimental data is insufficient for full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark data sets gave results that were comparable in quality with the results of multipolar refinement and superior to those for conventional models. Applications to several data sets of both small molecules and macromolecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package. text/html On macromolecular refinement at subatomic resolution with interatomic scatterers text 63 11 http://creativecommons.org/licenses/by/2.0/uk Acta Crystallographica Section D: Biological Crystallography 2007-11-01 1194 short communications 0907-4449 med@iucr.org 1197 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5113 This article focuses on the key step of obtaining the best possible sequence alignment of the Query (the protein you are interested in) to the Target (a protein of known three-dimensional structure) in order to build a molecular model for molecular replacement. Common sequence-alignment methods are discussed, starting from structural alignment and then moving to pairwise, multiple and profile–profile methods. The limitations of sequence-alignment methods and guidelines on how to judge the likely accuracy of alignment are considered. This is not a detailed tutorial on how to use specific programs; rather, the reader is directed to current tools and techniques that are likely to yield good results. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Barton, G.J. 2008-01-01 doi:10.1107/S0907444907046343 International Union of Crystallography The problems of gaining accurate protein sequence alignments for molecular replacement are discussed, current techniques explained and strategies suggested. en MOLECULAR REPLACEMENT; SEQUENCE ALIGNMENT This article focuses on the key step of obtaining the best possible sequence alignment of the Query (the protein you are interested in) to the Target (a protein of known three-dimensional structure) in order to build a molecular model for molecular replacement. Common sequence-alignment methods are discussed, starting from structural alignment and then moving to pairwise, multiple and profile–profile methods. The limitations of sequence-alignment methods and guidelines on how to judge the likely accuracy of alignment are considered. This is not a detailed tutorial on how to use specific programs; rather, the reader is directed to current tools and techniques that are likely to yield good results. text/html Sequence alignment for molecular replacement text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 25 research papers 0907-4449 med@iucr.org 32 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5105 An account is given of the latest developments of the AMoRe package: new rotational search algorithms, exploitation of noncrystallographic symmetry, generation and use of ensemble models and interactive graphical molecular replacement. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Trapani, S. Navaza, J. 2008-01-01 doi:10.1107/S0907444907044460 International Union of Crystallography An account is given of the latest developments of the AMoRe package. en AMORE; MOLECULAR REPLACEMENT An account is given of the latest developments of the AMoRe package: new rotational search algorithms, exploitation of noncrystallographic symmetry, generation and use of ensemble models and interactive graphical molecular replacement. text/html AMoRe: classical and modern text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 11 research papers 0907-4449 med@iucr.org 16 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5093 Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Stec, B. 2007-10-01 doi:10.1107/S0907444907041406 International Union of Crystallography Comment on Jaskolski et al. [(2007), Acta Cryst. D63, 611–620]. en STEREOCHEMICAL RESTRAINTS; REFINEMENT text/html Comment on Stereochemical restraints revisited: how accurate are refinement targets and how much should protein structures be allowed to deviate from them? by Jaskolski, Gilski, Dauter & Wlodawer (2007) text 63 10 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-10-01 1113 letters to the editor 0907-4449 med@iucr.org 1114 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5115 A novel automation pipeline for macromolecular structure solution by molecular replacement is described. There is a special emphasis on the discovery and preparation of a large number of search models, all of which can be passed to the core molecular-replacement programs. For routine molecular-replacement problems, the pipeline automates what a crystallographer might do and its value is simply one of convenience. For more difficult cases, the pipeline aims to discover the particular template structure and model edits required to produce a viable search model and may succeed in finding an efficacious combination that would be missed otherwise. An overview of MrBUMP is given and some recent additions to its functionality are highlighted. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Keegan, R.M. Winn, M.D. 2008-01-01 doi:10.1107/S0907444907037195 International Union of Crystallography An automation pipeline for macromolecular structure solution by molecular replacement with a special emphasis on the discovery and preparation of a large number of search models is described. en MOLECULAR REPLACEMENT; SEARCH-MODEL GENERATION; AUTOMATION; PROTEIN STRUCTURE A novel automation pipeline for macromolecular structure solution by molecular replacement is described. There is a special emphasis on the discovery and preparation of a large number of search models, all of which can be passed to the core molecular-replacement programs. For routine molecular-replacement problems, the pipeline automates what a crystallographer might do and its value is simply one of convenience. For more difficult cases, the pipeline aims to discover the particular template structure and model edits required to produce a viable search model and may succeed in finding an efficacious combination that would be missed otherwise. An overview of MrBUMP is given and some recent additions to its functionality are highlighted. text/html MrBUMP: an automated pipeline for molecular replacement text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 119 research papers 0907-4449 med@iucr.org 124 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5104 Molecular replacement is a powerful tool for the location of large models using structure-factor magnitudes alone. When phase information is available, it becomes possible to locate smaller fragments of the structure ranging in size from a few atoms to a single domain. The calculation is demanding, requiring a six-dimensional rotation and translation search. A number of approaches have been developed to this problem and a selection of these are reviewed in this paper. The application of one of these techniques to the problem of automated model building is explored in more detail, with particular reference to the problem of sequencing a protein main-chain trace. Copyright (c) 2008 International Union of Crystallography urn:issn:0907-4449 Cowtan, K. 2008-01-01 doi:10.1107/S0907444907033938 International Union of Crystallography A number of techniques for the location of small and medium-sized model fragments in experimentally phased electron-density maps are explored. The application of one of these techniques to automated model building is discussed. en MODEL FRAGMENTS; ELECTRON-DENSITY MAPS; MODEL BUILDING Molecular replacement is a powerful tool for the location of large models using structure-factor magnitudes alone. When phase information is available, it becomes possible to locate smaller fragments of the structure ranging in size from a few atoms to a single domain. The calculation is demanding, requiring a six-dimensional rotation and translation search. A number of approaches have been developed to this problem and a selection of these are reviewed in this paper. The application of one of these techniques to the problem of automated model building is explored in more detail, with particular reference to the problem of sequencing a protein main-chain trace. text/html Fitting molecular fragments into electron density text 64 1 Copyright (c) 2008 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2008-01-01 83 research papers 0907-4449 med@iucr.org 89 1399-0047 http://scripts.iucr.org/cgi-bin/paper?be5087 Proper solution of a macromolecular crystal structure based on anomalous scattering and/or isomorphous differences requires that the anomalous differences in reflection amplitudes be measured properly and that the correct enantiomer of the substructure be selected. If this information is wrong then the resulting electron-density maps will not show the correct structural features, but the reflection phases and map features will be related to the correct ones in a specific way. This text aims to explain how misinterpretation of the Bijvoet differences or of the substructure affects the resulting phases and electron-density maps. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Wang, J. Wlodawer, A. Dauter, Z. 2007-07-01 doi:10.1107/S0907444907025620 International Union of Crystallography The relationships between phases and electron-density maps obtained with correctly or wrongly interpreted anomalous differences or handedness of substructure are discussed. en PHASING; ANOMALOUS SCATTERING; SAD; INDEXING ERRORS Proper solution of a macromolecular crystal structure based on anomalous scattering and/or isomorphous differences requires that the anomalous differences in reflection amplitudes be measured properly and that the correct enantiomer of the substructure be selected. If this information is wrong then the resulting electron-density maps will not show the correct structural features, but the reflection phases and map features will be related to the correct ones in a specific way. This text aims to explain how misinterpretation of the Bijvoet differences or of the substructure affects the resulting phases and electron-density maps. text/html What happens when the signs of anomalous differences or the handedness of substructure are inverted? text 63 7 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-07-01 751 feature articles 0907-4449 med@iucr.org 758 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hm5049 The hairpin ribozyme is a small catalytic RNA comprising two helix–loop–helix domains linked by a four-way helical junction (4WJ). In its most basic form, each domain can be formed independently and reconstituted without a 4WJ to yield an active enzyme. The production of such minimal junctionless hairpin ribozymes is achievable by chemical synthesis, which has allowed structures to be determined for numerous nucleotide variants. However, abasic and other destabilizing core modifications hinder crystallization. This investigation describes the use of a dangling 5′-U to form an intermolecular U·U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L). Both linker constructs demonstrated similar enzymatic activity, but S9L constructs yielded crystals that diffracted to 2.65 Å resolution or better. In contrast, C3L variants diffracted to 3.35 Å and exhibited a 15 Å expansion of the c axis. Crystal packing of the C3L construct showed a paucity of 61 contacts, which comprise numerous backbone to 2′-OH hydrogen bonds in junctionless and S9L complexes. Significantly, the crystal packing in minimal structures mimics stabilizing features observed in the 4WJ hairpin ribozyme structure. The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 MacElrevey, C. Spitale, R.C. Krucinska, J. Wedekind, J.E. 2007-07-01 doi:10.1107/S090744490702464X International Union of Crystallography Insertion of a dangling 5′-uracil and incorporation of synthetic linkers at the domain interface of a minimal hairpin ribozyme have been investigated as means of favorably influencing crystal packing. These modifications lead to changes in the ribozyme's structural elements that mimic packing within a natural four-way helical junction, thereby providing an example of how knowledge-based design can be used to enhance the diffraction properties of a tertiarily folded RNA. en RNA; RIBOZYME; FOUR-WAY HELICAL JUNCTION; SYNTHETIC LINKER; CRYSTAL PACKING; STRUCTURE-BASED DESIGN The hairpin ribozyme is a small catalytic RNA comprising two helix–loop–helix domains linked by a four-way helical junction (4WJ). In its most basic form, each domain can be formed independently and reconstituted without a 4WJ to yield an active enzyme. The production of such minimal junctionless hairpin ribozymes is achievable by chemical synthesis, which has allowed structures to be determined for numerous nucleotide variants. However, abasic and other destabilizing core modifications hinder crystallization. This investigation describes the use of a dangling 5′-U to form an intermolecular U·U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L). Both linker constructs demonstrated similar enzymatic activity, but S9L constructs yielded crystals that diffracted to 2.65 Å resolution or better. In contrast, C3L variants diffracted to 3.35 Å and exhibited a 15 Å expansion of the c axis. Crystal packing of the C3L construct showed a paucity of 61 contacts, which comprise numerous backbone to 2′-OH hydrogen bonds in junctionless and S9L complexes. Significantly, the crystal packing in minimal structures mimics stabilizing features observed in the 4WJ hairpin ribozyme structure. The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects. text/html A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme text 63 7 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-07-01 812 research papers 0907-4449 med@iucr.org 825 1399-0047 http://scripts.iucr.org/cgi-bin/paper?wd5073 Automation of iterative model building, density modification and refinement in macromolecular crystallography has made it feasible to carry out this entire process multiple times. By using different random seeds in the process, a number of different models compatible with experimental data can be created. Sets of models were generated in this way using real data for ten protein structures from the Protein Data Bank and using synthetic data generated at various resolutions. Most of the heterogeneity among models produced in this way is in the side chains and loops on the protein surface. Possible interpretations of the variation among models created by repetitive rebuilding were investigated. Synthetic data were created in which a crystal structure was modelled as the average of a set of `perfect' structures and the range of models obtained by rebuilding a single starting model was examined. The standard deviations of coordinates in models obtained by repetitive rebuilding at high resolution are small, while those obtained for the same synthetic crystal structure at low resolution are large, so that the diversity within a group of models cannot generally be a quantitative reflection of the actual structures in a crystal. Instead, the group of structures obtained by repetitive rebuilding reflects the precision of the models, and the standard deviation of coordinates of these structures is a lower bound estimate of the uncertainty in coordinates of the individual models. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Terwilliger, T.C. Grosse-Kunstleve, R.W. Afonine, P.V. Adams, P.D. Moriarty, N.W. Zwart, P. Read, R.J. Turk, D. Hung, L.-W. 2007-05-01 doi:10.1107/S0907444907009791 International Union of Crystallography Heterogeneity in ensembles generated by independent model rebuilding principally reflects the limitations of the data and of the model-building process rather than the diversity of structures in the crystal. en MODEL BUILDING; MODEL COMPLETION; COORDINATE ERRORS; MODELS; PROTEIN DATA BANK; CONVERGENCE; REPRODUCIBILITY; HETEROGENEITY; PRECISION; ACCURACY Automation of iterative model building, density modification and refinement in macromolecular crystallography has made it feasible to carry out this entire process multiple times. By using different random seeds in the process, a number of different models compatible with experimental data can be created. Sets of models were generated in this way using real data for ten protein structures from the Protein Data Bank and using synthetic data generated at various resolutions. Most of the heterogeneity among models produced in this way is in the side chains and loops on the protein surface. Possible interpretations of the variation among models created by repetitive rebuilding were investigated. Synthetic data were created in which a crystal structure was modelled as the average of a set of `perfect' structures and the range of models obtained by rebuilding a single starting model was examined. The standard deviations of coordinates in models obtained by repetitive rebuilding at high resolution are small, while those obtained for the same synthetic crystal structure at low resolution are large, so that the diversity within a group of models cannot generally be a quantitative reflection of the actual structures in a crystal. Instead, the group of structures obtained by repetitive rebuilding reflects the precision of the models, and the standard deviation of coordinates of these structures is a lower bound estimate of the uncertainty in coordinates of the individual models. text/html Interpretation of ensembles created by multiple iterative rebuilding of macromolecular models text 63 5 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-05-01 597 research papers 0907-4449 med@iucr.org 610 1399-0047 http://scripts.iucr.org/cgi-bin/paper?dz0025 Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Wlodawer, A. 2007-03-01 doi:10.1107/S0907444907006336 International Union of Crystallography en STRUCTURAL DATA; DEPOSITION text/html Deposition of structural data redux text 63 3 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-03-01 421 letters to the editor 0907-4449 med@iucr.org 423 1399-0047 http://scripts.iucr.org/cgi-bin/paper?en5210 Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Muench, S.P. Prigge, S.T. McLeod, R. Rafferty, J.B. Kirisits, M.J. Roberts, C.W. Mui, E.J. Rice, D.W. 2007-03-01 doi:10.1107/S0907444906053625 International Union of Crystallography The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. en ENOYL ACYL CARRIER PROTEIN REDUCTASES; TRICLOSAN; APICOMPLEXAN PARASITES; PLASMODIUM FALCIPARUM; TOXOPLASMA GONDII Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials. text/html Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents text 63 3 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-03-01 328 research papers 0907-4449 med@iucr.org 338 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5103 In attempts to determine the crystal structure of small molecule–protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein–ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Chung, C. 2007-01-01 doi:10.1107/S0907444906051869 International Union of Crystallography This paper highlights some of the problems that can arise when attempting to obtain crystal structures of small molecule–protein complexes and how biophysical methods can be used to define and overcome these problems. Many of the techniques mentioned are also applicable to the study of protein–protein complexes and mode-of-action analysis. en PROTEIN-LIGAND COMPLEXES; ISOTHERMAL TITRATION CALORIMETRY; DYNAMIC LIGHT SCATTERING; NUCLEAR MAGNETIC RESONANCE; THERMAL DENATURATION In attempts to determine the crystal structure of small molecule–protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein–ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given. text/html The use of biophysical methods increases success in obtaining liganded crystal structures text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 62 research papers 0907-4449 med@iucr.org 71 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5074 The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 Å, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na+ sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Bento, I. Peixoto, C. Zaitsev, V.N. Lindley, P.F. 2007-02-01 doi:10.1107/S090744490604947X International Union of Crystallography The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. en CERULOPLASMIN; COPPER OXIDASES; METAL-BINDING SITES The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 Å, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na+ sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis. text/html Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites text 63 2 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-02-01 240 research papers 0907-4449 med@iucr.org 248 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5091 The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy (cryo-EM) single-particle reconstructions. Both techniques lean heavily on imposing icosahedral symmetry, thereby obscuring any deviation from the assumed symmetry. However, tailed bacteriophages have icosahedral or prolate icosahedral heads that have one obvious unique vertex where the genome can enter for DNA packaging and exit when infecting a host cell. The presence of the tail allows cryo-EM reconstructions in which the special vertex is used to orient the head in a unique manner. Some very large dsDNA icosahedral viruses also develop special vertices thought to be required for infecting host cells. Similarly, preliminary cryo-EM data for the small ssDNA canine parvovirus complexed with receptor suggests that these viruses, previously considered to be accurately icosahedral, might have some asymmetric properties that generate one preferred receptor-binding site on the viral surface. Comparisons are made between rhinoviruses that bind receptor molecules uniformly to all 60 equivalent binding sites, canine parvovirus, which appears to have a preferred receptor-binding site, and bacteriophage T4, which gains major biological advantages on account of its unique vertex and tail organelle. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Rossmann, M.G. Arisaka, F. Battisti, A.J. Bowman, V.D. Chipman, P.R. Fokine, A. Hafenstein, S. Kanamaru, S. Kostyuchenko, V.A. Mesyanzhinov, V.V. Shneider, M.M. Morais, M.C. Leiman, P.G. Palermo, L.M. Parrish, C.R. Xiao, C. 2007-01-01 doi:10.1107/S0907444906047330 International Union of Crystallography The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy single-particle reconstructions. This paper concerns itself with the study of the macromolecular complexes that constitute viruses, using structural hybrid techniques. en BACTERIOPHAGE T4; CANINE PARVOVIRUS; CRYO-ELECTRON MICROSCOPY; IMAGE RECONSTRUCTION; LARGE DSDNA ICOSAHEDRAL VIRUSES; SPECIAL VERTEX The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy (cryo-EM) single-particle reconstructions. Both techniques lean heavily on imposing icosahedral symmetry, thereby obscuring any deviation from the assumed symmetry. However, tailed bacteriophages have icosahedral or prolate icosahedral heads that have one obvious unique vertex where the genome can enter for DNA packaging and exit when infecting a host cell. The presence of the tail allows cryo-EM reconstructions in which the special vertex is used to orient the head in a unique manner. Some very large dsDNA icosahedral viruses also develop special vertices thought to be required for infecting host cells. Similarly, preliminary cryo-EM data for the small ssDNA canine parvovirus complexed with receptor suggests that these viruses, previously considered to be accurately icosahedral, might have some asymmetric properties that generate one preferred receptor-binding site on the viral surface. Comparisons are made between rhinoviruses that bind receptor molecules uniformly to all 60 equivalent binding sites, canine parvovirus, which appears to have a preferred receptor-binding site, and bacteriophage T4, which gains major biological advantages on account of its unique vertex and tail organelle. text/html From structure of the complex to understanding of the biology text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 9 research papers 0907-4449 med@iucr.org 16 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5102 Chronic myelogenous leukaemia (CML) results from the Bcr-Abl oncoprotein, which has a constitutively activated Abl tyrosine kinase domain. Although most chronic phase CML patients treated with imatinib as first-line therapy maintain excellent durable responses, patients who have progressed to advanced-stage CML frequently fail to respond or lose their response to therapy owing to the emergence of drug-resistant mutants of the protein. More than 40 such point mutations have been observed in imatinib-resistant patients. The crystal structures of wild-type and mutant Abl kinase in complex with imatinib and other small-molecule Abl inhibitors were determined, with the aim of understanding the molecular basis of resistance and to aid in the design and optimization of inhibitors active against the resistance mutants. These results are presented in a way which illustrates the approaches used to generate multiple structures, the type of information that can be gained and the way that this information is used to support drug discovery. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Cowan-Jacob, S.W. Fendrich, G. Floersheimer, A. Furet, P. Liebetanz, J. Rummel, G. Rheinberger, P. Centeleghe, M. Fabbro, D. Manley, P.W. 2007-01-01 doi:10.1107/S0907444906047287 International Union of Crystallography A case study showing how the determination of multiple cocrystal structures of the protein tyrosine kinase c-Abl was used to support drug discovery, resulting in a compound effective in the treatment of chronic myelogenous leukaemia. en TYROSINE KINASE; DRUG DISCOVERY; IMATINIB; NILOTINIB Chronic myelogenous leukaemia (CML) results from the Bcr-Abl oncoprotein, which has a constitutively activated Abl tyrosine kinase domain. Although most chronic phase CML patients treated with imatinib as first-line therapy maintain excellent durable responses, patients who have progressed to advanced-stage CML frequently fail to respond or lose their response to therapy owing to the emergence of drug-resistant mutants of the protein. More than 40 such point mutations have been observed in imatinib-resistant patients. The crystal structures of wild-type and mutant Abl kinase in complex with imatinib and other small-molecule Abl inhibitors were determined, with the aim of understanding the molecular basis of resistance and to aid in the design and optimization of inhibitors active against the resistance mutants. These results are presented in a way which illustrates the approaches used to generate multiple structures, the type of information that can be gained and the way that this information is used to support drug discovery. text/html Structural biology contributions to the discovery of drugs to treat chronic myelogenous leukaemia text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 80 research papers 0907-4449 med@iucr.org 93 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5100 Protein in crystal form is at an extremely high concentration and yet retains the complex secondary structure that defines an active protein. The protein crystal itself is made up of a repeating lattice of protein–protein and protein–solvent interactions. The problem that confronts any crystallographer is to identify those interactions that represent physiological interactions and those that do not. This review explores the tools that are available to provide such information using the original crystal liquor as a sample. The review is aimed at postgraduate and postdoctoral researchers who may well be coming up against this problem for the first time. Techniques are discussed that will provide information on the stoichiometry of complexes as well as low-resolution information on complex structure. Together, these data will help to identify the physiological complex. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Dafforn, T.R. 2007-01-01 doi:10.1107/S0907444906047044 International Union of Crystallography Structures of protein complexes offer some of the most interesting insights into biological processes. In this article, the methods required to show that the complex observed is the physiological one are investigated. en PROTEIN COMPLEX; SIZE-EXCLUSION CHROMATOGRAPHY; DYNAMIC LIGHT SCATTERING; ANALYTICAL ULTRACENTRIFUGATION; FLUORESCENCE RESONANCE ENERGY TRANSFER Protein in crystal form is at an extremely high concentration and yet retains the complex secondary structure that defines an active protein. The protein crystal itself is made up of a repeating lattice of protein–protein and protein–solvent interactions. The problem that confronts any crystallographer is to identify those interactions that represent physiological interactions and those that do not. This review explores the tools that are available to provide such information using the original crystal liquor as a sample. The review is aimed at postgraduate and postdoctoral researchers who may well be coming up against this problem for the first time. Techniques are discussed that will provide information on the stoichiometry of complexes as well as low-resolution information on complex structure. Together, these data will help to identify the physiological complex. text/html So how do you know you have a macromolecular complex? text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 17 research papers 0907-4449 med@iucr.org 25 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5097 Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Hassell, A.M. An, G. Bledsoe, R.K. Bynum, J.M. Carter, H.L. Deng, S.-J.J. Gampe, R.T. Grisard, T.E. Madauss, K.P. Nolte, R.T. Rocque, W.J. Wang, L. Weaver, K.L. Williams, S.P. Wisely, G.B. Xu, R. Shewchuk, L.M. 2007-01-01 doi:10.1107/S0907444906047020 International Union of Crystallography Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals. en PROTEIN-LIGAND COMPLEXES; CRYSTALLIZATION Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks. text/html Crystallization of protein–ligand complexes text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 72 research papers 0907-4449 med@iucr.org 79 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5101 Several potential applications of structural biology depend on discovering how one macromolecule might recognize a partner. Experiment remains the best way to answer this question, but computational tools can contribute where this fails. In such cases, structures may be studied to identify patches of exposed residues that have properties common to interaction surfaces and the locations of these patches can serve as the basis for further modelling or for further experimentation. To date, interaction surfaces have been proposed on the basis of unusual physical properties, unusual propensities for particular amino-acid types or an unusually high level of sequence conservation. Using the CXXSurface toolkit, developed as a part of the CCP4MG program, a suite of tools to analyse the properties of surfaces and their interfaces in complexes has been prepared and applied. These tools have enabled the rapid analysis of known complexes to evaluate the distribution of (i) hydrophobicity, (ii) electrostatic complementarity and (iii) sequence conservation in authentic complexes, so as to assess the extent to which these properties may be useful indicators of probable biological function. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Gruber, J. Zawaira, A. Saunders, R. Barrett, C.P. Noble, M.E.M. 2007-01-01 doi:10.1107/S0907444906046762 International Union of Crystallography This paper presents a survey of techniques that explore the surface properties of protein:protein interfaces so as to inform the prediction of probable sites of protein:protein interaction on newly determined protein structures. en SURFACES; ELECTROSTATICS; HYDROPHOBICITY; CONSERVATION Several potential applications of structural biology depend on discovering how one macromolecule might recognize a partner. Experiment remains the best way to answer this question, but computational tools can contribute where this fails. In such cases, structures may be studied to identify patches of exposed residues that have properties common to interaction surfaces and the locations of these patches can serve as the basis for further modelling or for further experimentation. To date, interaction surfaces have been proposed on the basis of unusual physical properties, unusual propensities for particular amino-acid types or an unusually high level of sequence conservation. Using the CXXSurface toolkit, developed as a part of the CCP4MG program, a suite of tools to analyse the properties of surfaces and their interfaces in complexes has been prepared and applied. These tools have enabled the rapid analysis of known complexes to evaluate the distribution of (i) hydrophobicity, (ii) electrostatic complementarity and (iii) sequence conservation in authentic complexes, so as to assess the extent to which these properties may be useful indicators of probable biological function. text/html Computational analyses of the surface properties of protein–protein interfaces text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 50 research papers 0907-4449 med@iucr.org 57 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5094 Isothermal titration calorimetry (ITC) provides highly complementary data to high-resolution structural detail. An overview of the methodology of the technique is provided. Ultimately, the correlation of the thermodynamic parameters determined by ITC with structural perturbation observed on going from the free to the bound state should be possible at an atomic level. Currently, thermodynamic data provide some insight as to potential changes occurring on complex formation. Here, this is demonstrated in the context of in vitro quantification of intracellular tyrosine kinase-mediated signal transduction and the issue of specificity of the important interactions. The apparent lack of specificity in the interactions of domains of proteins involved in early signalling from membrane-bound receptors is demonstrated using data from ITC. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Ladbury, J.E. 2007-01-01 doi:10.1107/S0907444906046373 International Union of Crystallography The use of isothermal titration calorimetry (ITC) provides a full thermodynamic characterization of an interaction in one experiment. The determination of the affinity is an important value; however, the additional layer of information provided by the change in enthalpy and entropy can help in understanding the biology. This is demonstrated with respect to tyrosine kinase-mediated signal transduction. en ISOTHERMAL TITRATION CALORIMETRY; FORMATION OF COMPLEXES; TYROSINE KINASE-MEDIATED SIGNAL TRANSDUCTION Isothermal titration calorimetry (ITC) provides highly complementary data to high-resolution structural detail. An overview of the methodology of the technique is provided. Ultimately, the correlation of the thermodynamic parameters determined by ITC with structural perturbation observed on going from the free to the bound state should be possible at an atomic level. Currently, thermodynamic data provide some insight as to potential changes occurring on complex formation. Here, this is demonstrated in the context of in vitro quantification of intracellular tyrosine kinase-mediated signal transduction and the issue of specificity of the important interactions. The apparent lack of specificity in the interactions of domains of proteins involved in early signalling from membrane-bound receptors is demonstrated using data from ITC. text/html Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 26 research papers 0907-4449 med@iucr.org 31 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5092 A procedure for the identification of ligands bound in crystal structures of macromolecules is described. Two characteristics of the density corresponding to a ligand are used in the identification procedure. One is the correlation of the ligand density with each of a set of test ligands after optimization of the fit of that ligand to the density. The other is the correlation of a fingerprint of the density with the fingerprint of model density for each possible ligand. The fingerprints consist of an ordered list of correlations of each the test ligands with the density. The two characteristics are scored using a Z-score approach in which the correlations are normalized to the mean and standard deviation of correlations found for a variety of mismatched ligand-density pairs, so that the Z scores are related to the probability of observing a particular value of the correlation by chance. The procedure was tested with a set of 200 of the most commonly found ligands in the Protein Data Bank, collectively representing 57% of all ligands in the Protein Data Bank. Using a combination of these two characteristics of ligand density, ranked lists of ligand identifications were made for representative (Fo − Fc)exp(iϕc) difference density from entries in the Protein Data Bank. In 48% of the 200 cases, the correct ligand was at the top of the ranked list of ligands. This approach may be useful in identification of unknown ligands in new macromolecular structures as well as in the identification of which ligands in a mixture have bound to a macromolecule. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Terwilliger, T.C. Adams, P.D. Moriarty, N.W. Cohn, J.D. 2007-01-01 doi:10.1107/S0907444906046233 International Union of Crystallography An automated ligand-fitting procedure is applied to (Fo − Fc)exp(iϕc) difference density for 200 commonly found ligands from macromolecular structures in the Protein Data Bank to identify ligands from density maps. en MODEL BUILDING; MODEL COMPLETION; SHAPE ANALYSIS A procedure for the identification of ligands bound in crystal structures of macromolecules is described. Two characteristics of the density corresponding to a ligand are used in the identification procedure. One is the correlation of the ligand density with each of a set of test ligands after optimization of the fit of that ligand to the density. The other is the correlation of a fingerprint of the density with the fingerprint of model density for each possible ligand. The fingerprints consist of an ordered list of correlations of each the test ligands with the density. The two characteristics are scored using a Z-score approach in which the correlations are normalized to the mean and standard deviation of correlations found for a variety of mismatched ligand-density pairs, so that the Z scores are related to the probability of observing a particular value of the correlation by chance. The procedure was tested with a set of 200 of the most commonly found ligands in the Protein Data Bank, collectively representing 57% of all ligands in the Protein Data Bank. Using a combination of these two characteristics of ligand density, ranked lists of ligand identifications were made for representative (Fo − Fc)exp(iϕc) difference density from entries in the Protein Data Bank. In 48% of the 200 cases, the correct ligand was at the top of the ranked list of ligands. This approach may be useful in identification of unknown ligands in new macromolecular structures as well as in the identification of which ligands in a mixture have bound to a macromolecule. text/html Ligand identification using electron-density map correlations text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 101 research papers 0907-4449 med@iucr.org 107 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5098 At the resolution available from most macromolecular crystals, the X-ray data alone are insufficient to lead to a chemically reasonable structure, so stereochemical restraints are essential. These usually restrain bond lengths, bond angles, planes and chiral volumes. The definition of these restraints and where the values come from are described. A dictionary entry contains information about the atom types, their connectivity and all the appropriate restraints. Torsion angles are not usually restrained, but they do have optimum values. In the special case of flexible five- and six-membered rings, including pentose and hexose sugars, the ring pucker is defined by combinations of torsion angles and the pucker affects the position of substituents. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Evans, P.R. 2007-01-01 doi:10.1107/S090744490604604X International Union of Crystallography A brief summary of the types of restraint defined in refinement dictionaries. en STEREOCHEMISTRY; RESTRAINTS; BOND LENGTHS; BOND ANGLES; PROTEIN STRUCTURE; CRYSTALLOGRAPHIC REFINEMENT At the resolution available from most macromolecular crystals, the X-ray data alone are insufficient to lead to a chemically reasonable structure, so stereochemical restraints are essential. These usually restrain bond lengths, bond angles, planes and chiral volumes. The definition of these restraints and where the values come from are described. A dictionary entry contains information about the atom types, their connectivity and all the appropriate restraints. Torsion angles are not usually restrained, but they do have optimum values. In the special case of flexible five- and six-membered rings, including pentose and hexose sugars, the ring pucker is defined by combinations of torsion angles and the pucker affects the position of substituents. text/html An introduction to stereochemical restraints text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 58 research papers 0907-4449 med@iucr.org 61 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5095 Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a `tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard `automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 McCoy, A.J. 2007-01-01 doi:10.1107/S0907444906045975 International Union of Crystallography Four case studies in using maximum-likelihood molecular replacement, as implemented in the program Phaser, to solve structures of protein complexes are described. en MACROMOLECULAR CRYSTALLOGRAPHY; MOLECULAR REPLACEMENT; MAXIMUM LIKELIHOOD Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a `tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard `automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases. text/html Solving structures of protein complexes by molecular replacement with Phaser text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 32 research papers 0907-4449 med@iucr.org 41 1399-0047 http://scripts.iucr.org/cgi-bin/paper?sx5064 Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and Rfree value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 Å diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Strop, P. Brzustowicz, M.R. Brunger, A.T. 2007-02-01 doi:10.1107/S0907444906045793 International Union of Crystallography An ab initio molecular-replacement method for phasing X-ray diffraction data for symmetric helical membrane proteins has been developed. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. en AB INITIO PHASING; MEMBRANE PROTEINS; MSCL; ION CHANNELS Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and Rfree value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 Å diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer. text/html Ab initio molecular-replacement phasing for symmetric helical membrane proteins text 63 2 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-02-01 188 research papers 0907-4449 med@iucr.org 196 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5073 The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual `reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Ciulli, A. Lobley, C.M.C. Tuck, K.L. Smith, A.G. Blundell, T.L. Abell, C. 2007-02-01 doi:10.1107/S0907444906044465 International Union of Crystallography A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. en BINDING MODES; CALORIMETRY; FRAGMENT-BASED DRUG DESIGN; KETOPANTOATE REDUCTASE; SITE-DIRECTED MUTAGENESIS The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual `reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes. text/html pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study text 63 2 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-02-01 171 research papers 0907-4449 med@iucr.org 178 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx9096 A correction is made to the name of one of the authors in Fogg et al. (2006), Acta Cryst. D62, 1196–1207. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Fogg, M.J. Alzari, P. Bahar, M. Bertini, I. Betton, J.-M. Burmeister, W.P. Cambillau, C. Canard, B. Carrondo, M.A. Coll, M. Daenke, S. Dym, O. Egloff, M.-P. Enguita, F.J. Geerlof, A. Haouz, A. Jones, T.A. Ma, Q. Manicka, S.N. Migliardi, M. Nordlund, P. Owens, R.J. Peleg, Y. Schneider, G. Schnell, R. Stuart, D.I. Tarbouriech, N. Unge, T. Wilkinson, A.J. Wilmanns, M. Wilson, K.S. Zimhony, O. Grimes, J.M. 2006-12-01 doi:10.1107/S0907444906044246 International Union of Crystallography A corrigendum to the paper by Fogg et al. (2006), Acta Cryst. D62, 1196–1207. en AUTOMATION; MINIATURIZATION; CLONING; EXPRESSION; VIRUSES; BACTERIA A correction is made to the name of one of the authors in Fogg et al. (2006), Acta Cryst. D62, 1196–1207. text/html Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens. Corrigendum text 62 12 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-12-01 1571 addenda and errata 0907-4449 med@iucr.org 1571 1399-0047 http://scripts.iucr.org/cgi-bin/paper?vr5063 The response regulator VicR from the Gram-positive bacterium Enterococcus faecalis forms part of the two-component signal transduction system of the YycFG subfamily. The structure of the DNA-binding domain of VicR, VicRc, has been solved and belongs to the winged helix–turn–helix family. It is very similar to the DNA-binding domains of Escherichia coli PhoB and OmpR, despite low sequence similarity, but differs in two important loops. The α-loop, which links the two helices of the helix–turn–helix motif, is similar to that of PhoB, where it has been implicated in contacting the σ subunit of RNA polymerase, but differs from that of OmpR. Conversely, the loop following the helix–turn–helix motif is similar to that of OmpR and differs from that of PhoB. YycF/VicR, PhoB and Bacillus subtilis PhoP regulators all recognize almost identical DNA sequences and although there is currently no experimental evidence linking this loop with the DNA, the structure is consistent with possible involvement in selective DNA recognition or binding. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Trinh, C.-H. Liu, Y. Phillips, S.E.V. Phillips-Jones, M.K. 2007-02-01 doi:10.1107/S0907444906043435 International Union of Crystallography The structure of the DNA-binding domain of the response regulator VicR from E. faecalis has been solved at 1.9 Å resolution. It is very similar to the related domains from PhoB and OmpR, but differs in two loops that may affect transcription activation or DNA–protein interactions. en RESPONSE REGULATOR; DNA-BINDING DOMAIN; TWO-COMPONENT SYSTEM; WINGED HELIX-TURN-HELIX The response regulator VicR from the Gram-positive bacterium Enterococcus faecalis forms part of the two-component signal transduction system of the YycFG subfamily. The structure of the DNA-binding domain of VicR, VicRc, has been solved and belongs to the winged helix–turn–helix family. It is very similar to the DNA-binding domains of Escherichia coli PhoB and OmpR, despite low sequence similarity, but differs in two important loops. The α-loop, which links the two helices of the helix–turn–helix motif, is similar to that of PhoB, where it has been implicated in contacting the σ subunit of RNA polymerase, but differs from that of OmpR. Conversely, the loop following the helix–turn–helix motif is similar to that of OmpR and differs from that of PhoB. YycF/VicR, PhoB and Bacillus subtilis PhoP regulators all recognize almost identical DNA sequences and although there is currently no experimental evidence linking this loop with the DNA, the structure is consistent with possible involvement in selective DNA recognition or binding. text/html Structure of the response regulator VicR DNA-binding domain text 63 2 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-02-01 266 short communications 0907-4449 med@iucr.org 269 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5093 Structural analysis of biological machines is essential for inferring their function and mechanism. Nevertheless, owing to their large size and instability, deciphering the atomic structure of macromolecular assemblies is still considered as a challenging task that cannot keep up with the rapid advances in the protein-identification process. In contrast, structural data at lower resolution is becoming more and more available owing to recent advances in cryo-electron microscopy (cryo-EM) techniques. Once a cryo-EM map is acquired, one of the basic questions asked is what are the folds of the components in the assembly and what is their configuration. Here, a novel knowledge-based computational method, named EMatch, towards tackling this task for cryo-EM maps at 6–10 Å resolution is presented. The method recognizes and locates possible atomic resolution structural homologues of protein domains in the assembly. The strengths of EMatch are demonstrated on a cryo-EM map of native GroEL at 6 Å resolution. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Dror, O. Lasker, K. Nussinov, R. Wolfson, H. 2007-01-01 doi:10.1107/S0907444906041059 International Union of Crystallography A method for detecting structural homologs of components in an intermediate resolution cryo-EM map and their spatial configuration is presented. en STRUCTURAL BIOINFORMATICS; INTERMEDIATE-RESOLUTION CRYO-EM MAPS; THREE-DIMENSIONAL ALIGNMENT OF SECONDARY STRUCTURES; MACROMOLECULAR ASSEMBLIES Structural analysis of biological machines is essential for inferring their function and mechanism. Nevertheless, owing to their large size and instability, deciphering the atomic structure of macromolecular assemblies is still considered as a challenging task that cannot keep up with the rapid advances in the protein-identification process. In contrast, structural data at lower resolution is becoming more and more available owing to recent advances in cryo-electron microscopy (cryo-EM) techniques. Once a cryo-EM map is acquired, one of the basic questions asked is what are the folds of the components in the assembly and what is their configuration. Here, a novel knowledge-based computational method, named EMatch, towards tackling this task for cryo-EM maps at 6–10 Å resolution is presented. The method recognizes and locates possible atomic resolution structural homologues of protein domains in the assembly. The strengths of EMatch are demonstrated on a cryo-EM map of native GroEL at 6 Å resolution. text/html EMatch: an efficient method for aligning atomic resolution subunits into intermediate-resolution cryo-EM maps of large macromolecular assemblies text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 42 research papers 0907-4449 med@iucr.org 49 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5070 Crystals of recombinant NovR (subunit MW = 29 924 Da; 270 amino acids), a non-haem iron oxygenase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group C2, with unit-cell parameters a = 86.69, b = 139.38, c = 100.82 Å, β = 101.18°. Native data were collected to a resolution of 2.1 Å from a single crystal at a synchrotron and a molecular-replacement solution was obtained using the program AMoRe. The starting phase information was very poor and did not permit model building. Phases were subsequently improved using a combination of fourfold averaging and very gradual phase extension in the program DM to yield an interpretable map. NovR belongs to a novel class of non-haem iron oxygenases that share sequence similarity with class II aldolases. It is predicted to perform two consecutive oxidative decarboxylation steps in the biosynthesis of the prenylated hydroxybenzoic acid moiety of the aminocoumarin antibiotic novobiocin. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Keller, S. Pojer, F. Heide, L. Lawson, D.M. 2006-12-01 doi:10.1107/S0907444906040169 International Union of Crystallography The crystal structure of NovR, a non-haem iron oxygenase from S. spheroides, was solved by molecular replacement with native X-ray data to 2.1 Å resolution using a template structure of relatively low sequence identity. An interpretable electron-density map was subsequently obtained from poor starting phases using a combination of fourfold averaging and very gradual phase extension. en NOVR; NON-HAEM IRON OXYGENASE; STREPTOMYCES; NOVOBIOCIN; ANTIBIOTIC BIOSYNTHESIS; MOLECULAR REPLACEMENT; DENSITY MODIFICATION; PHASE EXTENSION; LOCAL SYMMETRY AVERAGING Crystals of recombinant NovR (subunit MW = 29 924 Da; 270 amino acids), a non-haem iron oxygenase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group C2, with unit-cell parameters a = 86.69, b = 139.38, c = 100.82 Å, β = 101.18°. Native data were collected to a resolution of 2.1 Å from a single crystal at a synchrotron and a molecular-replacement solution was obtained using the program AMoRe. The starting phase information was very poor and did not permit model building. Phases were subsequently improved using a combination of fourfold averaging and very gradual phase extension in the program DM to yield an interpretable map. NovR belongs to a novel class of non-haem iron oxygenases that share sequence similarity with class II aldolases. It is predicted to perform two consecutive oxidative decarboxylation steps in the biosynthesis of the prenylated hydroxybenzoic acid moiety of the aminocoumarin antibiotic novobiocin. text/html Molecular replacement in the `twilight zone': structure determination of the non-haem iron oxygenase NovR from Streptomyces spheroides through repeated density modification of a poor molecular-replacement solution text 62 12 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-12-01 1564 short communications 0907-4449 med@iucr.org 1570 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5066 Eukaryotic aminoacyl-tRNA synthetases (aaRS) frequently contain additional appended domains that are absent from their prokaryotic counterparts which mediate complex formation between eukaryotic aaRS and cofactors of amino­acylation and translation. However, the structural basis of such interactions has remained elusive. The heteromerization domain of yeast glutamyl-tRNA synthetase (GluRS) has been cloned, expressed, purified and crystallized in space group C2221, with unit-cell parameters a = 52, b = 107, c = 168 Å. Phase information was obtained from multiple-wavelength anomalous dispersion with selenomethionine to 2.5 Å resolution and the structure, comprising two monomers per asymmetric unit, was determined and refined to 1.9 Å resolution. The structure of the interacting domain of its accessory protein Arc1p was determined and refined to 1.9 Å resolution in a crystal form containing 20 monomers organized in five tetramers per asymmetric unit (space group C2, unit-cell parameters a = 222, b = 89, c = 127 Å, β = 99.4°). Both domains adopt a GST-like fold, demonstrating a novel role for this fold as a protein–protein interaction module. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Simader, H. Hothorn, M. Suck, D. 2006-12-01 doi:10.1107/S0907444906039850 International Union of Crystallography The heteromerization domain of the glutamyl-tRNA synthetase (GluRS) from yeast was crystallized and phase information was obtained from selenomethionine MAD data to 2.5 Å resolution. This structure and that of the interacting domain of Arc1p, a protein cofactor of GluRS, were refined to 1.9 Å resolution. Both domains adopt a GST-like fold, demonstrating a novel role for this fold as a protein–protein interaction domain. en GLUTAMYL-TRNA SYNTHETASE; NUCLEAR-EXPORT COFACTOR ARC1P Eukaryotic aminoacyl-tRNA synthetases (aaRS) frequently contain additional appended domains that are absent from their prokaryotic counterparts which mediate complex formation between eukaryotic aaRS and cofactors of amino­acylation and translation. However, the structural basis of such interactions has remained elusive. The heteromerization domain of yeast glutamyl-tRNA synthetase (GluRS) has been cloned, expressed, purified and crystallized in space group C2221, with unit-cell parameters a = 52, b = 107, c = 168 Å. Phase information was obtained from multiple-wavelength anomalous dispersion with selenomethionine to 2.5 Å resolution and the structure, comprising two monomers per asymmetric unit, was determined and refined to 1.9 Å resolution. The structure of the interacting domain of its accessory protein Arc1p was determined and refined to 1.9 Å resolution in a crystal form containing 20 monomers organized in five tetramers per asymmetric unit (space group C2, unit-cell parameters a = 222, b = 89, c = 127 Å, β = 99.4°). Both domains adopt a GST-like fold, demonstrating a novel role for this fold as a protein–protein interaction module. text/html Structures of the interacting domains from yeast glutamyl-tRNA synthetase and tRNA-aminoacylation and nuclear-export cofactor Arc1p reveal a novel function for an old fold text 62 12 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-12-01 1510 research papers 0907-4449 med@iucr.org 1519 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5064 The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 Å resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-­hydroxy-3-­methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Leonard, P.M. Brzozowski, A.M. Lebedev, A. Marshall, C.M. Smith, D.J. Verma, C.S. Walton, N.J. Grogan, G. 2006-12-01 doi:10.1107/S0907444906039199 International Union of Crystallography The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL), the enzyme that catalyses the biotransformation of the coenzyme A thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] to vanillin (4-hydroxy-3-methoxy-benzaldehyde), has been solved and, in combination with a modelling study, a binding and discrimination mode for the substrate has been proposed. en VANILLIN; LYASE; CROTONASE; COENZYME A; HYDRATASE; RETRO-ALDOL The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 Å resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-­hydroxy-3-­methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate. text/html The 1.8 Å resolution structure of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL) from Pseudomonas fluorescens, an enzyme that catalyses the transformation of feruloyl-coenzyme A to vanillin text 62 12 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-12-01 1494 research papers 0907-4449 med@iucr.org 1501 1399-0047 http://scripts.iucr.org/cgi-bin/paper?hv5063 Mycobacterium tuberculosis is a facultative intracellular parasite of alveolar macrophages. M. tuberculosis is able to propagate in harsh environments within cells such as phagocytes, despite being exposed to reactive oxygen and nitrogen intermediates. The thioredoxin redox system is conserved across the phyla and has a well characterized role in resisting oxidative stress and influencing gene expression within prokaryotic and eukaryotic cells. M. tuberculosis thioredoxin (MtbTrx) has similar functions in redox homeostasis and it has recently been shown that alkyl hydroperoxidase C is efficiently reduced to its active form by MtbTrxC, supporting this notion. To address whether the MtbTrx has similar features to other thioredoxin structures and to examine the opportunities for designing drugs against this target, MtbTrxC has been crystallized and its structure determined to 1.3 Å resolution. Unexpectedly, the structure demonstrates an interesting crystal packing in which five C-­terminal residues from the MtbTrxC fold insert into a groove adjacent to the active site. A very similar interaction is observed in structures of human thioredoxins bound to peptides from the target proteins NF-κB and Ref-1. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Hall, G. Shah, M. McEwan, P.A. Laughton, C. Stevens, M. Westwell, A. Emsley, J. 2006-12-01 doi:10.1107/S0907444906038212 International Union of Crystallography The 1.3 Å resolution crystal structure of M. tuberculosis thioredoxin C is reported, demonstrating a novel packing of five C-terminal residues in the active-site groove. en MYCOBACTERIUM TUBERCULOSIS; THIOREDOXINS; PEROXIDASES Mycobacterium tuberculosis is a facultative intracellular parasite of alveolar macrophages. M. tuberculosis is able to propagate in harsh environments within cells such as phagocytes, despite being exposed to reactive oxygen and nitrogen intermediates. The thioredoxin redox system is conserved across the phyla and has a well characterized role in resisting oxidative stress and influencing gene expression within prokaryotic and eukaryotic cells. M. tuberculosis thioredoxin (MtbTrx) has similar functions in redox homeostasis and it has recently been shown that alkyl hydroperoxidase C is efficiently reduced to its active form by MtbTrxC, supporting this notion. To address whether the MtbTrx has similar features to other thioredoxin structures and to examine the opportunities for designing drugs against this target, MtbTrxC has been crystallized and its structure determined to 1.3 Å resolution. Unexpectedly, the structure demonstrates an interesting crystal packing in which five C-­terminal residues from the MtbTrxC fold insert into a groove adjacent to the active site. A very similar interaction is observed in structures of human thioredoxins bound to peptides from the target proteins NF-κB and Ref-1. text/html Structure of Mycobacterium tuberculosis thioredoxin C text 62 12 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-12-01 1453 research papers 0907-4449 med@iucr.org 1457 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ol5293 Gaucher disease is caused by mutations in the gene encoding acid-β-glucosidase. A recombinant form of this enzyme, Cerezyme®, is used to treat Gaucher disease patients by `enzyme-replacement therapy'. Crystals of Cerezyme® after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 Å resolution [Dvir et al. (2003), EMBO Rep. 4, 704–709]. The crystal structure of unmodified Cerezyme® is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme® is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-β-­glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Brumshtein, B. Wormald, M.R. Silman, I. Futerman, A.H. Sussman, J.L. 2006-12-01 doi:10.1107/S0907444906038303 International Union of Crystallography Crystals of native Cerezyme® (acid-β-glucosidase), the enzyme used in enzyme-replacement therapy in Gaucher disease, were obtained and the threee-dimensional structure solved. A number of sugar residues bound to three asparagines via N-glycosylation could be observed in the structure, and new conoformations of loops controlling access to the active site were detected. en GAUCHER DISEASE; GLUCOSIDASES; CEREZYME[REGISTERED SIGN]; ENZYME-REPLACEMENT THERAPY; GLYCANS; XYLANASES Gaucher disease is caused by mutations in the gene encoding acid-β-glucosidase. A recombinant form of this enzyme, Cerezyme®, is used to treat Gaucher disease patients by `enzyme-replacement therapy'. Crystals of Cerezyme® after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 Å resolution [Dvir et al. (2003), EMBO Rep. 4, 704–709]. The crystal structure of unmodified Cerezyme® is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme® is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-β-­glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes. text/html Structural comparison of differently glycosylated forms of acid-β-glucosidase, the defective enzyme in Gaucher disease text 62 12 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-12-01 1458 research papers 0907-4449 med@iucr.org 1465 1399-0047 http://scripts.iucr.org/cgi-bin/paper?ba5090 Crystal structures deposited in the Protein Data Bank illustrate the diversity of biological macromolecular recognition: transient interactions in protein–protein and protein–DNA complexes and permanent assemblies in homodimeric proteins. The geometric and physical chemical properties of the macromolecular interfaces that may govern the stability and specificity of recognition are explored in complexes and homodimers compared with crystal-packing interactions. It is found that crystal-packing interfaces are usually much smaller; they bury fewer atoms and are less tightly packed than in specific assemblies. Standard-size interfaces burying 1200–2000 Å2 of protein surface occur in protease–inhibitor and antigen–antibody complexes that assemble with little or no conformation changes. Short-lived electron-transfer complexes have small interfaces; the larger size of the interfaces observed in complexes involved in signal transduction and homodimers correlates with the presence of conformation changes, often implicated in biological function. Results of the CAPRI (critical assessment of predicted interactions) blind prediction experiment show that docking algorithms efficiently and accurately predict the mode of assembly of proteins that do not change conformation when they associate. They perform less well in the presence of large conformation changes and the experiment stimulates the development of novel procedures that can handle such changes. Copyright (c) 2007 International Union of Crystallography urn:issn:0907-4449 Janin, J. Rodier, F. Chakrabarti, P. Bahadur, R.P. 2007-01-01 doi:10.1107/S090744490603575X International Union of Crystallography X-ray structures in the PDB illustrate both the specific recognition of two polypeptide chains in protein–protein complexes and dimeric proteins and their nonspecific interaction at crystal contacts. en MACROMOLECULAR RECOGNITION; PROTEIN DATA BANK Crystal structures deposited in the Protein Data Bank illustrate the diversity of biological macromolecular recognition: transient interactions in protein–protein and protein–DNA complexes and permanent assemblies in homodimeric proteins. The geometric and physical chemical properties of the macromolecular interfaces that may govern the stability and specificity of recognition are explored in complexes and homodimers compared with crystal-packing interactions. It is found that crystal-packing interfaces are usually much smaller; they bury fewer atoms and are less tightly packed than in specific assemblies. Standard-size interfaces burying 1200–2000 Å2 of protein surface occur in protease–inhibitor and antigen–antibody complexes that assemble with little or no conformation changes. Short-lived electron-transfer complexes have small interfaces; the larger size of the interfaces observed in complexes involved in signal transduction and homodimers correlates with the presence of conformation changes, often implicated in biological function. Results of the CAPRI (critical assessment of predicted interactions) blind prediction experiment show that docking algorithms efficiently and accurately predict the mode of assembly of proteins that do not change conformation when they associate. They perform less well in the presence of large conformation changes and the experiment stimulates the development of novel procedures that can handle such changes. text/html Macromolecular recognition in the Protein Data Bank text 63 1 Copyright (c) 2007 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2007-01-01 1 research papers 0907-4449 med@iucr.org 8 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx5087 As part of the Structural Proteomics In Europe (SPINE) project, the automated multifermenter system GRETA has been developed with structural genomics as a major application. GRETA comprises 6–24 parallel fermentation chambers, each with individual control of fermentation parameters such as temperature, stirring, pH, dissolved oxygen concentration and feed profiles. Six human proteins were used to optimize the GRETA fermentation processes and to compare these processes with typical baffled-flask protocols used in structural genomics projects. The optimized GRETA processes allows several times more protein to be produced per litre of culture with limited manual intervention and constitutes a potentially useful alternative both for scale-up production in structural proteomic projects and for fermentation-process optimization. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Hedrén, M. Ballagi, A. Mörtsell, L. Rajkai, G. Stenmark, P. Sturesson, C. Nordlund, P. 2006-10-01 doi:10.1107/S090744490603441X International Union of Crystallography A multifermentation system, GRETA, has been developed which specifically accommodates features that respond to the needs of future structural genomics programmes. The GRETA multifermenter provides a robust platform for streamlined parallel fermentation, requiring minimal manual intervention, while at the same time providing a versatile fermentation platform for process optimization. en STRUCTURAL GENOMICS FERMENTATION; HIGH CELL DENSITY; HIGH-THROUGHPUT PIPELINE As part of the Structural Proteomics In Europe (SPINE) project, the automated multifermenter system GRETA has been developed with structural genomics as a major application. GRETA comprises 6–24 parallel fermentation chambers, each with individual control of fermentation parameters such as temperature, stirring, pH, dissolved oxygen concentration and feed profiles. Six human proteins were used to optimize the GRETA fermentation processes and to compare these processes with typical baffled-flask protocols used in structural genomics projects. The optimized GRETA processes allows several times more protein to be produced per litre of culture with limited manual intervention and constitutes a potentially useful alternative both for scale-up production in structural proteomic projects and for fermentation-process optimization. text/html GRETA, a new multifermenter system for structural genomics and process optimization text 62 10 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-10-01 1227 research papers 0907-4449 med@iucr.org 1231 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx5095 A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining `low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Au, K. Berrow, N.S. Blagova, E. Boucher, I.W. Boyle, M.P. Brannigan, J.A. Carter, L.G. Dierks, T. Folkers, G. Grenha, R. Harlos, K. Kaptein, R. Kalliomaa, A.K. Levdikov, V.M. Meier, C. Milioti, N. Moroz, O. Müller, A. Owens, R.J. Rzechorzek, N. Sainsbury, S. Stuart, D.I. Walter, T.S. Waterman, D.G. Wilkinson, A.J. Wilson, K.S. Zaccai, N. Esnouf, R.M. Fogg, M.J. 2006-10-01 doi:10.1107/S0907444906033555 International Union of Crystallography The current state of high-throughput technologies for protein production and structure determination is assessed in relation to progress on the SPINE structural proteomics project on proteins from B. anthracis. en TARGET SELECTION; LIGATION-INDEPENDENT CLONING; PROTEIN EXPRESSION; HIGH THROUGHPUT; NANOLITRE-SCALE CRYSTALLIZATION; AUTOMATED STRUCTURE DETERMINATION; ANTHRAX A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining `low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs. text/html Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis text 62 10 Copyright (c) 2006 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 2006-10-01 1267 research papers 0907-4449 med@iucr.org 1275 1399-0047 http://scripts.iucr.org/cgi-bin/paper?gx5090 Not all proteins form well defined three-dimensional structures in their native states. Some amino-acid sequences appear to strongly favour the disordered state, whereas some can apparently transition between disordered and ordered states under the influence of changes in the biological environment, thereby playing an important role in processes such as signalling. Although important biologically, for the structural biologist disordered regions of proteins can be disastrous even preventing successful structure determination. The accurate prediction of disorder is therefore important, not least for directing the design of expression constructs so as to maximize the chances of successful structure determination. Such design criteria have become integral to the construct-design strategies of laboratories within the Structural Proteomics In Europe (SPINE) consortium. This paper assesses the current state of the art in disorder prediction in terms of prediction reliability and considers how best to use these methods to guide construct design. Finally, it presents a brief discussion as to how methods of prediction might be improved in the future. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Esnouf, R.M. Hamer, R. Sussman, J.L. Silman, I. Trudgian, D. Yang, Z.-R. Prilusky, J. 2006-10-01 doi:10.1107/S0907444906033580 International Union of Crystallography The current state of methods for the in silico prediction of natively disordered regions of proteins is discussed in the context of the potential for improvement and application of these predictions in structural proteomics. en STRUCTURAL PROTEOMICS; NATIVELY UNFOLDED PROTEIN; INTRINSICALLY DISORDERED PROTEIN; PROTEIN CRYSTALLIZATION; HIGH THROUGHPUT; BIOINFORMATICS Not all proteins form well defined three-dimensional structures in their native states. Some amino-acid sequences appear to strongly favour the disordered state, whereas some can apparently transition between disordered and ordered states under the influence of changes in the biological env