http://journals.iucr.org/d/issues/2009/12/00/isscontsbdy.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 papers on crystallographic binding studies, structural analysis of mutants and other structure-function studies. Refinements of previously known structures may be published if sufficient new information is presented. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. en Copyright (c) 2009 International Union of Crystallography 2009-12-01 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 papers on crystallographic binding studies, structural analysis of mutants and other structure-function studies. Refinements of previously known structures may be published if sufficient new information is presented. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. text/html Acta Crystallographica Section D: Biological Crystallography, Volume 65, Part 12, 2009 text monthly 1 2002-01-01T00:00+00:00 12 65 2009-12-01 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 1237 urn:issn:0907-4449 med@iucr.org December 2009 2009-12-01 http://journals.iucr.org/logos/rss10d.gif http://journals.iucr.org/d/issues/2009/12/00/isscontsbdy.html Still image http://scripts.iucr.org/cgi-bin/paper?gm5010 Dehydration of protein crystals is rarely used, despite being a post-crystallization method that is useful for the improvement of crystal diffraction properties, as it is difficult to reproduce and monitor. A novel device for hydration control of macromolecular crystals in a standard data-collection environment has been developed. The device delivers an air stream of precise relative humidity that can be used to alter the amount of water in macromolecular crystals. The device can be rapidly installed and is fully compatible with most standard synchrotron X-ray beamlines. Samples are mounted in cryoloops and the progress of dehydration can be monitored both optically and by the acquisition of diffraction images. Once the optimal hydration level has been obtained, cryocooling is easy to achieve by hand or by using a sample changer. The device has been thoroughly tested on several ESRF beamlines and is available to users. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Sanchez-Weatherby, J. Bowler, M.W. Huet, J. Gobbo, A. Felisaz, F. Lavault, B. Moya, R. Kadlec, J. Ravelli, R.B.G. Cipriani, F. 2009-11-17 doi:10.1107/S0907444909037822 International Union of Crystallography The HC1b is a new user-friendly humidity-control device that is compatible with standard X-ray beamlines. It is used to improve the diffraction properties of macromolecular crystals by optimizing their hydration state. en humidity-control device X-ray diffraction improvement crystallographic synchrotron instrumentation crystal dehydration Dehydration of protein crystals is rarely used, despite being a post-crystallization method that is useful for the improvement of crystal diffraction properties, as it is difficult to reproduce and monitor. A novel device for hydration control of macromolecular crystals in a standard data-collection environment has been developed. The device delivers an air stream of precise relative humidity that can be used to alter the amount of water in macromolecular crystals. The device can be rapidly installed and is fully compatible with most standard synchrotron X-ray beamlines. Samples are mounted in cryoloops and the progress of dehydration can be monitored both optically and by the acquisition of diffraction images. Once the optimal hydration level has been obtained, cryocooling is easy to achieve by hand or by using a sample changer. The device has been thoroughly tested on several ESRF beamlines and is available to users. text/html Improving diffraction by humidity control: a novel device compatible with X-ray beamlines text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1237 1246 http://scripts.iucr.org/cgi-bin/paper?en5379 α-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P41212, with unit-cell parameters a = b = 42.231, c = 93.655 Å, and that diffracts to 1.45 Å resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Cámara-Artigas, A. Andújar-Sánchez, M. Ortiz-Salmerón, E. Cuadri, C. Casares, S. 2009-11-17 doi:10.1107/S0907444909038037 International Union of Crystallography The key role of Pro20 in the crystal nucleation and growth rate of the α-spectrin SH3 domain has been investigated. en SH3 domains nucleation crystallization mechanism α-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P41212, with unit-cell parameters a = b = 42.231, c = 93.655 Å, and that diffracts to 1.45 Å resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth. text/html The effect of a proline residue on the rate of growth and the space group of α-spectrin SH3-domain crystals text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1247 1252 http://scripts.iucr.org/cgi-bin/paper?mv5029 Bacterial l-asparaginases have been used in the treatment of childhood acute lymphoblastic leukaemia for over 30 years. Their therapeutic effect is based on their ability to catalyze the conversion of l-asparagine, an essential amino acid in certain tumours, to l-aspartic acid and ammonia. Two l-asparaginases, one from Escherichia coli and the other from Erwinia chrysanthemi, have been widely employed in clinical practice as anti-leukaemia drugs. However, l-asparaginases are also able to cause severe side effects owing to their intrinsic glutaminase activity. Helicobacter pylori l-asparaginase (HpA) has been reported to have negligible glutaminase activity. To gain insight into the properties of HpA, its crystal structure in the presence of l-aspartate was determined to 1.4 Å resolution, which is one of the highest resolutions obtained for an l-asparaginase structure. The final structure has an Rcryst of 12.6% (Rfree = 16.9%) with good stereochemistry. A detailed analysis of the active site showed major differences in the active-site flexible loop and in the 286–297 loop from the second subunit, which is involved in active-site formation. Accordingly, Glu289, Asn255 and Gln63 are suggested to play roles in modulating the accessibility of the active site. Overall, the structural comparison revealed that HpA has greater structural similarity to E. coli l-asparaginase than to any other l-asparaginase, including Er. carotovora l-asparaginase, despite the fact that the latter is also characterized by low glutaminase activity. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Dhavala, P. Papageorgiou, A.C. 2009-11-17 doi:10.1107/S0907444909038244 International Union of Crystallography The crystal structure of H. pylori l-asparaginase in the presence of l-Asp was determined at 1.4 Å resolution and revealed changes in loops involved in the formation of the active site. en l-asparaginases active site leukaemia treatment therapeutic enzymes Bacterial l-asparaginases have been used in the treatment of childhood acute lymphoblastic leukaemia for over 30 years. Their therapeutic effect is based on their ability to catalyze the conversion of l-asparagine, an essential amino acid in certain tumours, to l-aspartic acid and ammonia. Two l-asparaginases, one from Escherichia coli and the other from Erwinia chrysanthemi, have been widely employed in clinical practice as anti-leukaemia drugs. However, l-asparaginases are also able to cause severe side effects owing to their intrinsic glutaminase activity. Helicobacter pylori l-asparaginase (HpA) has been reported to have negligible glutaminase activity. To gain insight into the properties of HpA, its crystal structure in the presence of l-aspartate was determined to 1.4 Å resolution, which is one of the highest resolutions obtained for an l-asparaginase structure. The final structure has an Rcryst of 12.6% (Rfree = 16.9%) with good stereochemistry. A detailed analysis of the active site showed major differences in the active-site flexible loop and in the 286–297 loop from the second subunit, which is involved in active-site formation. Accordingly, Glu289, Asn255 and Gln63 are suggested to play roles in modulating the accessibility of the active site. Overall, the structural comparison revealed that HpA has greater structural similarity to E. coli l-asparaginase than to any other l-asparaginase, including Er. carotovora l-asparaginase, despite the fact that the latter is also characterized by low glutaminase activity. text/html Structure of Helicobacter pyloril-asparaginase at 1.4 Å resolution text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1253 1261 http://scripts.iucr.org/cgi-bin/paper?mv5030 Adhesion/invasion of pathogenic bacteria is a critical step in infection and is mediated by surface-exposed proteins termed adhesins. The crystal structure of recombinant Lmb, a laminin-binding adhesin from Streptococcus agalactiae, has been determined at 2.5 Å resolution. Based on sequence and structural homology, Lmb was placed into the cluster 9 family of the ABC (ATP-binding cassette) transport system. The structural organization of Lmb closely resembles that of ABC-type solute-binding proteins (SBPs), in which two structurally related globular domains interact with each other to form a metal-binding cavity at the interface. The bound zinc in Lmb is tetrahedrally coordinated by three histidines and a glutamate from both domains. A comparison of Lmb with other metal transporters revealed an interesting feature of the dimerization of molecules in the crystallographic asymmetric unit in all zinc-binding transporters. A closer comparison of Lmb with the zinc-binding ZnuA from Escherichia coli and Synechocystis 6803 suggested that Lmb might undergo a unique structural rearrangement upon metal binding and release. The crystal structure of Lmb provides an impetus for further investigations into the molecular basis of laminin binding by human pathogens. Being ubiquitous in all serotypes of group B streptococcus (GBS), the structure of Lmb may direct the development of an efficient vaccine. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Ragunathan, P. Spellerberg, B. Ponnuraj, K. 2009-11-17 doi:10.1107/S0907444909038359 International Union of Crystallography The crystal structure of a laminin-binding adhesin (Lmb) from S. agalactiae was determined at 2.5 Å resolution. The structure of Lmb will allow further investigations into the possible dual role of Lmb as an adhesin and in metal homeostasis. en adhesins metal transporters surface proteins laminin binding ATP-binding cassette solute-binding proteins Adhesion/invasion of pathogenic bacteria is a critical step in infection and is mediated by surface-exposed proteins termed adhesins. The crystal structure of recombinant Lmb, a laminin-binding adhesin from Streptococcus agalactiae, has been determined at 2.5 Å resolution. Based on sequence and structural homology, Lmb was placed into the cluster 9 family of the ABC (ATP-binding cassette) transport system. The structural organization of Lmb closely resembles that of ABC-type solute-binding proteins (SBPs), in which two structurally related globular domains interact with each other to form a metal-binding cavity at the interface. The bound zinc in Lmb is tetrahedrally coordinated by three histidines and a glutamate from both domains. A comparison of Lmb with other metal transporters revealed an interesting feature of the dimerization of molecules in the crystallographic asymmetric unit in all zinc-binding transporters. A closer comparison of Lmb with the zinc-binding ZnuA from Escherichia coli and Synechocystis 6803 suggested that Lmb might undergo a unique structural rearrangement upon metal binding and release. The crystal structure of Lmb provides an impetus for further investigations into the molecular basis of laminin binding by human pathogens. Being ubiquitous in all serotypes of group B streptococcus (GBS), the structure of Lmb may direct the development of an efficient vaccine. text/html Structure of laminin-binding adhesin (Lmb) from Streptococcus agalactiae text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1262 1269 http://scripts.iucr.org/cgi-bin/paper?bw5305 A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 Å resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 McLellan, T.J. Marr, E.S. Wondrack, L.M. Subashi, T.A. Aeed, P.A. Han, S. Xu, Z. Wang, I.-K. Maguire, B.A. 2009-11-17 doi:10.1107/S0907444909039043 International Union of Crystallography A description is given of a robust procedure for the purification of 50S ribosomal subunits from D. radiodurans that consistently crystallize to form crystals that give diffraction patterns extending to 2.9 Å resolution. Factors that may influence the ability to form crystals are systematically evaluated and future areas of study aresuggested. en ribosome 50S subunit crystallization purification mass spectrometry Deinococcus radiodurans A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 Å resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested. text/html A systematic study of 50S ribosomal subunit purification enabling robust crystallization text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1270 1282 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. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Urzhumtsev, A. Afonine, P.V. Adams, P.D. 2009-11-17 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 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1283 1291 http://scripts.iucr.org/cgi-bin/paper?dz5170 The structure of the X (or ADRP) domain of a pathogenic variant of feline coronavirus (FCoV) has been determined in tetragonal and cubic crystal forms to 3.1 and 2.2 Å resolution, respectively. In the tetragonal crystal form, glycerol-3-phosphate was observed in the ADP-ribose-binding site. Both crystal forms contained large solvent channels and had a solvent content of higher than 70%. Only very weak binding of this domain to ADP-ribose was detected in vitro. However, the structure with ADP-ribose bound was determined in the cubic crystal form at 3.9 Å resolution. The structure of the FCoV X domain had the expected macro-domain fold and is the first structure of this domain from a coronavirus belonging to subgroup 1a. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Wojdyla, J.A. Manolaridis, I. Snijder, E.J. Gorbalenya, A.E. Coutard, B. Piotrowski, Y. Hilgenfeld, R. Tucker, P.A. 2009-11-17 doi:10.1107/S0907444909040074 International Union of Crystallography The structure of the X (ADRP) domain of nonstructural protein 3 from feline coronavirus has been determined in two crystal forms. The binding of glycerol-3-phosphate and ADP-ribose is described. en feline coronavirus X domains ADRP domains nonstructural protein 3 The structure of the X (or ADRP) domain of a pathogenic variant of feline coronavirus (FCoV) has been determined in tetragonal and cubic crystal forms to 3.1 and 2.2 Å resolution, respectively. In the tetragonal crystal form, glycerol-3-phosphate was observed in the ADP-ribose-binding site. Both crystal forms contained large solvent channels and had a solvent content of higher than 70%. Only very weak binding of this domain to ADP-ribose was detected in vitro. However, the structure with ADP-ribose bound was determined in the cubic crystal form at 3.9 Å resolution. The structure of the FCoV X domain had the expected macro-domain fold and is the first structure of this domain from a coronavirus belonging to subgroup 1a. text/html Structure of the X (ADRP) domain of nsp3 from feline coronavirus text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1292 1300 http://scripts.iucr.org/cgi-bin/paper?gx5155 CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723–1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813–1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Miller, M. Dauter, Z. Cherry, S. Tropea, J.E. Wlodawer, A. 2009-11-17 doi:10.1107/S0907444909040153 International Union of Crystallography The crystal structure of the Taz2 zinc-finger domain of the human p300 transcriptional coactivator was determined using the anomalous diffraction signal of the bound Zn ions. Crystal contacts suggested a possible novel mode of Taz2–peptide ligand interactions. en zinc-finger proteins anomalous diffraction protein recognition transcription regulation CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723–1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813–1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed. text/html Structure of the Taz2 domain of p300: insights into ligand binding text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1301 1308 http://scripts.iucr.org/cgi-bin/paper?hm5078 Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis–Menten kinetics, with a Km of 60.7 mM and a kcat of 21.7 s−1. The structure of the enzyme was solved at a resolution of 1.9 Å in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite −1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite −1 from the `nonreducing' end. Binding of the glucosyl moiety in subsite −1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the `nonreducing' end during the glucan-elongation cycle. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Champion, E. Remaud-Simeon, M. Skov, L.K. Kastrup, J.S. Gajhede, M. Mirza, O. 2009-11-17 doi:10.1107/S0907444909040311 International Union of Crystallography The crystal structure of apo sucrose hydrolase from X. campestris pv. campestris is reported. en glycoside hydrolase family 13 sucrose hydrolysis active-site conformation reaction mechanism Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis–Menten kinetics, with a Km of 60.7 mM and a kcat of 21.7 s−1. The structure of the enzyme was solved at a resolution of 1.9 Å in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite −1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite −1 from the `nonreducing' end. Binding of the glucosyl moiety in subsite −1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the `nonreducing' end during the glucan-elongation cycle. text/html The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1309 1314 http://scripts.iucr.org/cgi-bin/paper?dz5173 NAD+-dependent formate dehydrogenase (FDH) catalyzes the oxidation of formate ion to carbon dioxide coupled with the reduction of NAD+ to NADH. The crystal structures of the apo and holo forms of FDH from the methylotrophic bacterium Moraxella sp. C-1 (MorFDH) are reported at 1.96 and 1.95 Å resolution, respectively. MorFDH is similar to the previously studied FDH from the bacterium Pseudomonas sp. 101 in overall structure, cofactor-binding mode and active-site architecture, but differs in that the eight-residue-longer C-terminal fragment is visible in the electron-density maps of MorFDH. MorFDH also differs in the organization of the dimer interface. The holo MorFDH structure supports the earlier hypothesis that the catalytic residue His332 can form a hydrogen bond to both the substrate and the transition state. Apo MorFDH has a closed conformation of the interdomain cleft, which is unique for an apo form of an NAD+-dependent dehydrogenase. A comparison of the structures of bacterial FDH in open and closed conformations allows the differentiation of the conformational changes associated with cofactor binding and domain motion and provides insights into the mechanism of the closure of the interdomain cleft in FDH. The C-terminal residues 374–399 and the substrate (formate ion) or inhibitor (azide ion) binding are shown to play an essential role in the transition from the open to the closed conformation. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Shabalin, I.G. Filippova, E.V. Polyakov, K.M. Sadykhov, E.G. Safonova, T.N. Tikhonova, T.V. Tishkov, V.I. Popov, V.O. 2009-11-17 doi:10.1107/S0907444909040773 International Union of Crystallography The crystal structures of the apo and holo forms of NAD+-dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Moraxella sp. C-1 are reported. The structure of the apo form in the closed conformation provides insights into the mechanism of closure of the interdomain cleft in FDH. en NAD+-dependent formate dehydrogenases protein conformation interdomain cleft dimer interface apo and holo forms NAD+-dependent formate dehydrogenase (FDH) catalyzes the oxidation of formate ion to carbon dioxide coupled with the reduction of NAD+ to NADH. The crystal structures of the apo and holo forms of FDH from the methylotrophic bacterium Moraxella sp. C-1 (MorFDH) are reported at 1.96 and 1.95 Å resolution, respectively. MorFDH is similar to the previously studied FDH from the bacterium Pseudomonas sp. 101 in overall structure, cofactor-binding mode and active-site architecture, but differs in that the eight-residue-longer C-terminal fragment is visible in the electron-density maps of MorFDH. MorFDH also differs in the organization of the dimer interface. The holo MorFDH structure supports the earlier hypothesis that the catalytic residue His332 can form a hydrogen bond to both the substrate and the transition state. Apo MorFDH has a closed conformation of the interdomain cleft, which is unique for an apo form of an NAD+-dependent dehydrogenase. A comparison of the structures of bacterial FDH in open and closed conformations allows the differentiation of the conformational changes associated with cofactor binding and domain motion and provides insights into the mechanism of the closure of the interdomain cleft in FDH. The C-terminal residues 374–399 and the substrate (formate ion) or inhibitor (azide ion) binding are shown to play an essential role in the transition from the open to the closed conformation. text/html Structures of the apo and holo forms of formate dehydrogenase from the bacterium Moraxella sp. C-1: towards understanding the mechanism of the closure of the interdomain cleft text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1315 1325 http://scripts.iucr.org/cgi-bin/paper?mh5029 The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 Å resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (α-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg2+ ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Watanabe, N. Takasaki, Y. Sato, C. Ando, S. Tanaka, I. 2009-11-17 doi:10.1107/S0907444909041134 International Union of Crystallography The crystal structure of the HindIII–cognate DNA complex was solved with and without metal cations. en restriction endonucleases DNA complex vaporizing-iodine labelling method The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 Å resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (α-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg2+ ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism. text/html Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 1326 1333 http://scripts.iucr.org/cgi-bin/paper?dz5174 Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: am = bo − co, bm = ao and cm = −2co − bo. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Radaev, S. Agniswamy, J. Sun, P.D. 2009-11-17 doi:10.1107/S0907444909039912 International Union of Crystallography When properly applied, pseudosymmetry can be used to improve crystallographic phases through averaging and to facilitate crystal structure determination. en pseudosymmetry structure determination cysteine proteases Mac-1 Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: am = bo − co, bm = ao and cm = −2co − bo. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement. text/html A case of structure determination using pseudosymmetry text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography short communications 1334 1340 http://scripts.iucr.org/cgi-bin/paper?be5137 Some of the current trends in the structure of evolutionary biology are reviewed using as a framework the book of the same title by S. J. Gould (1941–2002). The revised concepts and interpretations of the structure–function relationship in evolutionary biology are discussed in relation to the past achievements and future developments in structural biology. Copyright (c) 2009 International Union of Crystallography urn:issn:0907-4449 Abad-Zapatero, C. 2009-11-17 doi:10.1107/S0907444909044370 International Union of Crystallography The new developments in evolutionary biology are reviewed to encourage a fruitful dialog between structural and evolutionary biologists. en Evolutionary theory evolution of protein structure evolution of molecular structure adaptationism evolutionary constraints Some of the current trends in the structure of evolutionary biology are reviewed using as a framework the book of the same title by S. J. Gould (1941–2002). The revised concepts and interpretations of the structure–function relationship in evolutionary biology are discussed in relation to the past achievements and future developments in structural biology. text/html Notes of a protein crystallographer: the molecular structure of evolutionary theory text 12 65 2009-11-17 Copyright (c) 2009 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography essays 1341 1349