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) 2008 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) 2008 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?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 64 863 2008-08-01 Acta Crystallographica Section D: Biological Crystallography research papers 851 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 64 904 2008-08-01 Acta Crystallographica Section D: Biological Crystallography short communications 902 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 64 729 2008-07-01 Acta Crystallographica Section D: Biological Crystallography research papers 711 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 short communications 700 704 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 short communications 607 610 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 research papers 515 524 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 research papers 525 531 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 research papers 487 495 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 letters to the editor 335 336 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 research papers 416 424 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 research papers 377 382 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 research papers 158 166 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 research papers 99 107 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 research papers 108 118 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 research papers 76 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 research papers 90 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 research papers 40 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 research papers 70 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 research papers 1 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 research papers 61 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 letters to the editor 1274 1281 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 research papers 125 132 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 research papers 133 140 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 research papers 1208 1216 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 research papers 17 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 research papers 33 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 letters to the editor 1282 1283 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 research papers 49 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. Comm