http://journals.iucr.org/d/journalhomepage.html Acta Crystallographica Section D: Biological Crystallography welcomes submission of papers covering any aspect of biological crystallography, particularly structures of biological macromolecules. In addition to new structural determinations, preliminary data on unit-cell dimensions and space groups will be considered for publication, provided suitable diffraction photographs (or their equivalent), together with an estimate of resolution, are included. Also, articles on crystal growth of biological macromolecules are welcomed, and refinements of known structures may be published if the information content warrants it. en-gb Copyright (c) 2009 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 submission of papers covering any aspect of biological crystallography, particularly structures of biological macromolecules. In addition to new structural determinations, preliminary data on unit-cell dimensions and space groups will be considered for publication, provided suitable diffraction photographs (or their equivalent), together with an estimate of resolution, are included. Also, articles on crystal growth of biological macromolecules are welcomed, and refinements of known structures may be published if the information content warrants it. 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) 2009 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?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 10 65 2009-10-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 1062 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 10 65 2009-10-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 1051 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 9 65 2009-09-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 letters to the editor 1009 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 9 65 2009-09-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 952 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 9 65 2009-09-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 932 med@iucr.org 941 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 9 65 2009-09-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 letters to the editor 1007 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 9 65 2009-09-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 913 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 9 65 2009-09-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 900 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 690 med@iucr.org 696 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 8 65 2009-08-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 839 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 8 65 2009-08-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 786 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 8 65 2009-08-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 758 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 679 med@iucr.org 689 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 6 65 2009-06-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 582 med@iucr.org 601 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 672 med@iucr.org 678 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 6 65 2009-06-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 602 med@iucr.org 610 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 659 med@iucr.org 671 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 6 65 2009-06-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 553 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 633 med@iucr.org 643 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 644 med@iucr.org 650 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 6 65 2009-06-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 513 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]. Copyright (c) 2009 International Union of Crystallography 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 3 65 2009-03-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 addenda and errata 301 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 651 med@iucr.org 658 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 7 65 2009-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2009 International Union of Crystallography 0907-4449 research papers 625 med@iucr.org 632 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 4 65 2009-04-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 short communications 379 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 156 med@iucr.org 168 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 193 med@iucr.org 199 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 101 med@iucr.org 111 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 134 med@iucr.org 139 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 128 med@iucr.org 133 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 3 65 2009-03-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 241 med@iucr.org 248 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 148 med@iucr.org 155 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 140 med@iucr.org 147 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 3 65 2009-03-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 201 med@iucr.org 211 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 121 med@iucr.org 127 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 112 med@iucr.org 120 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 176 med@iucr.org 185 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 186 med@iucr.org 192 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 12 64 2008-12-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 1187 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 11 64 2008-11-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 1116 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 9 64 2008-09-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 letters to the editor 1000 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 9 64 2008-09-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 957 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 2 65 2009-02-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 169 med@iucr.org 175 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 8 64 2008-08-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 874 med@iucr.org 882 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 8 64 2008-08-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 851 med@iucr.org 863 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 8 64 2008-08-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 short communications 902 med@iucr.org 904 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 7 64 2008-07-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 711 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 6 64 2008-06-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 short communications 700 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 6 64 2008-06-01 Acta Crystallographica Section D: Biological Crystallography 0907-4449 research papers 611 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 5 64 2008-05-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 short communications 607 med@iucr.org 610 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 5 64 2008-05-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 515 med@iucr.org 524 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 5 64 2008-05-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 525 med@iucr.org 531 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 5 64 2008-05-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 487 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 3 64 2008-03-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 letters to the editor 335 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 4 64 2008-04-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 416 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 4 64 2008-04-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 377 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 2 64 2008-02-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 158 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 99 med@iucr.org 107 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 108 med@iucr.org 118 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 76 med@iucr.org 82 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 90 med@iucr.org 98 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 40 med@iucr.org 48 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 70 med@iucr.org 75 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 1 med@iucr.org 10 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 61 med@iucr.org 69 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 12 63 2007-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 letters to the editor 1274 med@iucr.org 1281 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 125 med@iucr.org 132 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 133 med@iucr.org 140 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 12 63 2007-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 1208 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 17 med@iucr.org 24 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 33 med@iucr.org 39 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 12 63 2007-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 letters to the editor 1282 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 49 med@iucr.org 60 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 25 med@iucr.org 32 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 11 med@iucr.org 16 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 10 63 2007-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 letters to the editor 1113 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 119 med@iucr.org 124 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 1 64 2008-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2008 International Union of Crystallography 0907-4449 research papers 83 med@iucr.org 89 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 7 63 2007-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 feature articles 751 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 7 63 2007-07-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 812 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 5 63 2007-05-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 597 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 3 63 2007-03-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 letters to the editor 421 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 3 63 2007-03-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 328 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 62 med@iucr.org 71 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 2 63 2007-02-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 240 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 9 med@iucr.org 16 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 80 med@iucr.org 93 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 17 med@iucr.org 25 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 72 med@iucr.org 79 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 50 med@iucr.org 57 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 26 med@iucr.org 31 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 101 med@iucr.org 107 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 32 med@iucr.org 41 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 58 med@iucr.org 61 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 2 63 2007-02-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 188 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 2 63 2007-02-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 171 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 12 62 2006-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 addenda and errata 1571 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 2 63 2007-02-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 short communications 266 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 42 med@iucr.org 49 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 12 62 2006-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 short communications 1564 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 12 62 2006-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1510 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 12 62 2006-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1494 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 12 62 2006-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1453 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 12 62 2006-12-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1458 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 1 63 2007-01-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2007 International Union of Crystallography 0907-4449 research papers 1 med@iucr.org 8 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 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1227 med@iucr.org 1231 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 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. text/html Honing the in silico toolkit for detecting protein disorder text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1260 med@iucr.org 1266 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 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1267 med@iucr.org 1275 http://scripts.iucr.org/cgi-bin/paper?gx5077 An automatic data-collection system has been implemented and installed on seven insertion-device beamlines and a bending-magnet beamline at the ESRF (European Synchrotron Radiation Facility) as part of the SPINE (Structural Proteomics In Europe) development of an automated structure-determination pipeline. The system allows remote interaction with beamline-control systems and automatic sample mounting, alignment, characterization, data collection and processing. Reports of all actions taken are available for inspection via database modules and web services. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Beteva, A. Cipriani, F. Cusack, S. Delageniere, S. Gabadinho, J. Gordon, E.J. Guijarro, M. Hall, D.R. Larsen, S. Launer, L. Lavault, C.B. Leonard, G.A. Mairs, T. McCarthy, A. McCarthy, J. Meyer, J. Mitchell, E. Monaco, S. Nurizzo, D. Pernot, P. Pieritz, R. Ravelli, R.G.B. Rey, V. Shepard, W. Spruce, D. Stuart, D.I. Svensson, O. Theveneau, P. Thibault, X. Turkenburg, J. Walsh, M. McSweeney, S.M. 2006-10-01 doi:10.1107/S0907444906032859 International Union of Crystallography Automated data collection for macromolecular crystallography at beamlines with protocols for automatic instrumentation improves the efficient use of available beamtime. These developments were a result of a collaboration between the main partners of SPINE and members of other similar European and national initiatives. en AUTOMATION; DATA-COLLECTION PIPELINE; LIMS An automatic data-collection system has been implemented and installed on seven insertion-device beamlines and a bending-magnet beamline at the ESRF (European Synchrotron Radiation Facility) as part of the SPINE (Structural Proteomics In Europe) development of an automated structure-determination pipeline. The system allows remote interaction with beamline-control systems and automatic sample mounting, alignment, characterization, data collection and processing. Reports of all actions taken are available for inspection via database modules and web services. text/html High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1162 med@iucr.org 1169 http://scripts.iucr.org/cgi-bin/paper?gx5089 This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless 13C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 AB, E. Atkinson, A.R. Banci, L. Bertini, I. Ciofi-Baffoni, S. Brunner, K. Diercks, T. Dötsch, V. Engelke, F. Folkers, G.E. Griesinger, C. Gronwald, W. Günther, U. Habeck, M. de Jong, R.N. Kalbitzer, H.R. Kieffer, B. Leeflang, B.R. Loss, S. Luchinat, C. Marquardsen, T. Moskau, D. Neidig, K.-P. Nilges, M. Piccioli, M. Pierattelli, R. Rieping, W. Schippmann, T. Schwalbe, H. Travé, G. Trenner, J. Wöhnert, J. Zweckstetter, M. Kaptein, R. 2006-10-01 doi:10.1107/S0907444906032070 International Union of Crystallography New hardware and software developments for protein structure determination by NMR are described. These techniques allow faster, more automated solution-structure determination and more efficient data utilization. en NMR SPECTROSCOPY; HIGH THROUGHPUT; INTERFERENTIAL STRUCTURE DETERMINATIONS; PROTONLESS 13C-DIRECT DETECTION METHODS This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless 13C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations. text/html NMR in the SPINE Structural Proteomics project text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1150 med@iucr.org 1161 http://scripts.iucr.org/cgi-bin/paper?gx5093 The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Bahar, M. Ballard, C. Cohen, S.X. Cowtan, K.D. Dodson, E.J. Emsley, P. Esnouf, R.M. Keegan, R. Lamzin, V. Langer, G. Levdikov, V. Long, F. Meier, C. Muller, A. Murshudov, G.N. Perrakis, A. Siebold, C. Stein, N. Turkenburg, M.G.W. Vagin, A.A. Winn, M. Winter, G. Wilson, K.S. 2006-10-01 doi:10.1107/S0907444906032197 International Union of Crystallography The results of a workshop on the automation of protein crystal structure solution organized by the Structural Proteomics In Europe (SPINE) consortium are reported. en AUTOMATION; MOLECULAR REPLACEMENT; DATA QUALITY; MODEL-BUILDING TOOLS The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level. text/html SPINE workshop on automated X-ray analysis: a progress report text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1170 med@iucr.org 1183 http://scripts.iucr.org/cgi-bin/paper?gx5098 Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His6-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore `expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Berrow, N.S. Büssow, K. Coutard, B. Diprose, J. Ekberg, M. Folkers, G.E. Levy, N. Lieu, V. Owens, R.J. Peleg, Y. Pinaglia, C. Quevillon-Cheruel, S. Salim, L. Scheich, C. Vincentelli, R. Busso, D. 2006-10-01 doi:10.1107/S0907444906031337 International Union of Crystallography This article presents a comparative analysis of the standard methods for small-scale screening of recombinant protein expression/solubility implemented in laboratories of the Structural Proteomics In Europe (SPINE) consortium. en PROTEIN EXPRESSION; PROTEIN PRODUCTION; HIGH THROUGHPUT Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His6-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore `expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization. text/html Recombinant protein expression and solubility screening in Escherichia coli: a comparative study text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1218 med@iucr.org 1226 http://scripts.iucr.org/cgi-bin/paper?gx5096 The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (∼220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens. 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. 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. 2006-10-01 doi:10.1107/S0907444906030915 International Union of Crystallography Progress by the SPINE consortium on the determination of structures of proteins of bacterial and viral pathogens is outlined, with particular reference to the impact of high-throughput technologies. en AUTOMATION; MINIATURIZATION; CLONING; EXPRESSION; VIRUSES; BACTERIA The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (∼220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens. text/html Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1196 med@iucr.org 1207 http://scripts.iucr.org/cgi-bin/paper?gx5080 Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression tech­niques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Romier, C. Ben Jelloul, M. Albeck, S. Buchwald, G. Busso, D. Celie, P.H.N. Christodoulou, E. De Marco, V. van Gerwen, S. Knipscheer, P. Lebbink, J.H. Notenboom, V. Poterszman, A. Rochel, N. Cohen, S.X. Unger, T. Sussman, J.L. Moras, D. Sixma, T.K. Perrakis, A. 2006-10-01 doi:10.1107/S0907444906031003 International Union of Crystallography The design of a new system for multi-expression in E. coli and a database for co-expression data are described. Lessons are drawn from case studies using these and other strategies for co-expression and analysis of protein complexes. en PROTEIN CO-EXPRESSION; HIGH THROUGHPUT; MULTI-EXPRESSION SYSTEM Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression tech­niques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells. text/html Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1232 med@iucr.org 1242 http://scripts.iucr.org/cgi-bin/paper?gx5085 A standard sample holder and vial for cryocooled macromolecular crystals has been defined for use with robotic sample changers. This SPINE standard sample holder is a modified version, with added features and specifications, of sample holders in common use. In particular, the SPINE standard meets the precision required for automatic sample exchange and includes a cap that is identified by a two-dimensional datamatrix code as well as an optional vial. At the ESRF, the sample holder standard is in use with the EMBL/ESRF/BM14 robotic sample changer (SC3) which is installed on eight beamlines. The SC3 can hold up to 50 crystals stored in five baskets. A datamatrix reader in the SC3 ensures safe management of the sample flow and facilitates fully automatic screening and characterization of samples. Tools for handling and transporting 50 samples in a dry shipping dewar have been developed. In addition to the SC3, the SPINE sample holder is currently compatible with a number of other robotic sample changers. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Cipriani, F. Felisaz, F. Launer, L. Aksoy, J.-S. Caserotto, H. Cusack, S. Dallery, M. di-Chiaro, F. Guijarro, M. Huet, J. Larsen, S. Lentini, M. McCarthy, J. McSweeney, S. Ravelli, R. Renier, M. Taffut, C. Thompson, A. Leonard, G.A. Walsh, M.A. 2006-10-01 doi:10.1107/S0907444906030587 International Union of Crystallography A sample holder standard for use with robotic sample changers is defined. The standard includes a system for sample identification, tracking and management of data flow in a macromolecular structure-determination pipeline. A robotic sample changer designed for use with the sample standard is described. en SPINE SAMPLE HOLDER STANDARD; DATAMATRIX IDENTIFICATION; BEAMLINE AUTOMATION; ROBOTIC SAMPLE CHANGER; LIMS; CRYO CRYSTAL MOUNT A standard sample holder and vial for cryocooled macromolecular crystals has been defined for use with robotic sample changers. This SPINE standard sample holder is a modified version, with added features and specifications, of sample holders in common use. In particular, the SPINE standard meets the precision required for automatic sample exchange and includes a cap that is identified by a two-dimensional datamatrix code as well as an optional vial. At the ESRF, the sample holder standard is in use with the EMBL/ESRF/BM14 robotic sample changer (SC3) which is installed on eight beamlines. The SC3 can hold up to 50 crystals stored in five baskets. A datamatrix reader in the SC3 ensures safe management of the sample flow and facilitates fully automatic screening and characterization of samples. Tools for handling and transporting 50 samples in a dry shipping dewar have been developed. In addition to the SC3, the SPINE sample holder is currently compatible with a number of other robotic sample changers. text/html Automation of sample mounting for macromolecular crystallography text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1251 med@iucr.org 1259 http://scripts.iucr.org/cgi-bin/paper?gx5084 Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein–protein and protein–ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Geerlof, A. Brown, J. Coutard, B. Egloff, M.-P. Enguita, F.J. Fogg, M.J. Gilbert, R.J.C. Groves, M.R. Haouz, A. Nettleship, J.E. Nordlund, P. Owens, R.J. Ruff, M. Sainsbury, S. Svergun, D.I. Wilmanns, M. 2006-10-01 doi:10.1107/S0907444906030307 International Union of Crystallography Characteristics such as the purity of target proteins are key factors that can influence the success of structure determination. Here, the application and efficacy of various quality-assessment methodologies are discussed. en PROTEIN CHARACTERIZATION; STRUCTURAL PROTEOMICS; QUALITY ASSESSMENT Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein–protein and protein–ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given. text/html The impact of protein characterization in structural proteomics text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1125 med@iucr.org 1136 http://scripts.iucr.org/cgi-bin/paper?gx5078 Epstein–Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known enzyme activity and then supplementing this set based on a series of predicted properties, in particular secondary structure. The major obstacle turned out to be poor expression and low solubility. Surprisingly, this could not be overcome by modifications of the constructs, changes of expression temperature or strain or renaturation. Of the eight soluble proteins, five were crystallized using robotic nanolitre-drop crystallization trials, which led to four solved structures. Although these results depended on individual treatment rather than standardized protocols, a high-throughput miniaturized crystallization screening protocol was a key component of success with these difficult proteins. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Tarbouriech, N. Buisson, M. Géoui, T. Daenke, S. Cusack, S. Burmeister, W.P. 2006-10-01 doi:10.1107/S0907444906030034 International Union of Crystallography For a structural genomics project on Epstein–Barr virus, 23 proteins were cloned into E. coli for protein expression. The structures of four have been solved by X-ray crystallography. en STRUCTURAL GENOMICS; EBV; EPSTEIN-BARR VIRUS; GENOME ANNOTATION; PROTEIN EXPRESSION Epstein–Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known enzyme activity and then supplementing this set based on a series of predicted properties, in particular secondary structure. The major obstacle turned out to be poor expression and low solubility. Surprisingly, this could not be overcome by modifications of the constructs, changes of expression temperature or strain or renaturation. Of the eight soluble proteins, five were crystallized using robotic nanolitre-drop crystallization trials, which led to four solved structures. Although these results depended on individual treatment rather than standardized protocols, a high-throughput miniaturized crystallization screening protocol was a key component of success with these difficult proteins. text/html Structural genomics of the Epstein–Barr virus text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1276 med@iucr.org 1285 http://scripts.iucr.org/cgi-bin/paper?gx5081 The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Alzari, P.M. Berglund, H. Berrow, N.S. Blagova, E. Busso, D. Cambillau, C. Campanacci, V. Christodoulou, E. Eiler, S. Fogg, M.J. Folkers, G. Geerlof, A. Hart, D. Haouz, A. Herman, M.D. Macieira, S. Nordlund, P. Perrakis, A. Quevillon-Cheruel, S. Tarandeau, F. van Tilbeurgh, H. Unger, T. Luna-Vargas, M.P.A. Velarde, M. Willmanns, M. Owens, R.J. 2006-10-01 doi:10.1107/S0907444906029775 International Union of Crystallography The technology output from the Structural Proteomics In Europe (SPINE) consortium for cloning and expression in E. coli is reviewed. en CLONING; EXPRESSION SCREENING The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation. text/html Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1103 med@iucr.org 1113 http://scripts.iucr.org/cgi-bin/paper?gx5099 The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Aricescu, A.R. Assenberg, R. Bill, R.M. Busso, D. Chang, V.T. Davis, S.J. Dubrovsky, A. Gustafsson, L. Hedfalk, K. Heinemann, U. Jones, I.M. Ksiazek, D. Lang, C. Maskos, K. Messerschmidt, A. Macieira, S. Peleg, Y. Perrakis, A. Poterszman, A. Schneider, G. Sixma, T.K. Sussman, J.L. Sutton, G. Tarboureich, N. Zeev-Ben-Mordehai, T. Jones, E.Y. 2006-10-01 doi:10.1107/S0907444906029805 International Union of Crystallography Strategies and experience resulting from the use of eukaryotic expression systems in SPINE are described. en HIGH THROUGHPUT; EUKARYOTIC EXPRESSION; BACULOVIRUS; YEAST; MAMMALIAN CELLS The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed. text/html Eukaryotic expression: developments for structural proteomics text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1114 med@iucr.org 1124 http://scripts.iucr.org/cgi-bin/paper?gx5082 SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Albeck, S. Alzari, P. Andreini, C. Banci, L. Berry, I.M. Bertini, I. Cambillau, C. Canard, B. Carter, L. Cohen, S.X. Diprose, J.M. Dym, O. Esnouf, R.M. Felder, C. Ferron, F. Guillemot, F. Hamer, R. Ben Jelloul, M. Laskowski, R.A. Laurent, T. Longhi, S. Lopez, R. Luchinat, C. Malet, H. Mochel, T. Morris, R.J. Moulinier, L. Oinn, T. Pajon, A. Peleg, Y. Perrakis, A. Poch, O. Prilusky, J. Rachedi, A. Ripp, R. Rosato, A. Silman, I. Stuart, D.I. Sussman, J.L. Thierry, J.-C. Thompson, J.D. Thornton, J.M. Unger, T. Vaughan, B. Vranken, W. Watson, J.D. Whamond, G. Henrick, K. 2006-10-01 doi:10.1107/S090744490602991X International Union of Crystallography This article reviews the infrastructure of software that has been developed under the SPINE initiative for high-throughput protein-structure analysis covering target selection and prototype LIMS for protein production through to structure annotation, with contributions to the management of a large-scale crystallization set-up and structure determination. en STRUCTURAL GENOMICS; HIGH THROUGHPUT; TARGET SELECTION; STRUCTURE ANNOTATION; DATA MANAGEMENT SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited. text/html SPINE bioinformatics and data-management aspects of high-throughput structural biology text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1184 med@iucr.org 1195 http://scripts.iucr.org/cgi-bin/paper?gx5088 Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, pro­kary­otic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Aricescu, A.R. Lu, W. Jones, E.Y. 2006-10-01 doi:10.1107/S0907444906029799 International Union of Crystallography A simple, fast and affordable method for transient protein expression in mammalian cells is presented. It combines several features of interest for X-ray crystallography such as high protein yield, straightforward purification, selenomethinonine incorporation and control of N-linked glycosylation. en EUKARYOTIC EXPRESSION; MAMMALIAN CELLS; SELENOMETHIONINE; PROTEIN PRODUCTION Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, pro­kary­otic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline. text/html A time- and cost-efficient system for high-level protein production in mammalian cells text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1243 med@iucr.org 1250 http://scripts.iucr.org/cgi-bin/paper?gx5086 This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Berry, I.M. Dym, O. Esnouf, R.M. Harlos, K. Meged, R. Perrakis, A. Sussman, J.L. Walter, T.S. Wilson, J. Messerschmidt, A. 2006-10-01 doi:10.1107/S090744490602943X International Union of Crystallography The introduction of automation and minimization into the crystallization pipeline has improved the yield of high-quality crystals for structural determination using more reproducible experimental protocols. en CRYSTALLIZATION; AUTOMATION; MINIMIZATION This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future. text/html SPINE high-throughput crystallization, crystal imaging and recognition techniques: current state, performance analysis, new technologies and future aspects text 10 62 2006-10-01 Acta Crystallographica Section D: Biological Crystallography Copyright (c) 2006 International Union of Crystallography 0907-4449 research papers 1137 med@iucr.org 1149 http://scripts.iucr.org/cgi-bin/paper?gx5083 The EC `Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ∼170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples. Copyright (c) 2006 International Union of Crystallography urn:issn:0907-4449 Banci, L. Bertini, I. Cusack, S. de Jong, R.N. Heinemann, U. Jones, E.Y. Kozielski, F. Maskos, K. Messerschmidt, A. Owens, R. Perrakis, A. Poterszman, A. Schneider, G. Siebold, C. Silman, I. Sixma, T. Stewart-Jones, G.