[Show abstract][Hide abstract] ABSTRACT: Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition process, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2005; 61(Pt 9):818-20. DOI:10.1107/S1744309105026126 · 0.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.
Proteins Structure Function and Bioinformatics 10/2005; 60(4):787-96. DOI:10.1002/prot.20541 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.
[Show abstract][Hide abstract] ABSTRACT: The computer programs ARP/wARP, MAID and RESOLVE are designed to build protein structures into experimentally phased electron-density maps without any user intervention, requiring only diffraction data and sequence information. However, the MAID and RESOLVE systems, which seek to extend the range of automated model-building to approximately 3 A resolution, have yet to receive significant testing outside the small numbers of data sets used in their development. Since these two systems employ a large number of scoring functions and decision-making heuristics, additional tests are required to establish their usefulness to the crystallographic community. To independently evaluate these programs, their performance was tested using a database containing 41 experimentally phased maps between 1.3 and 2.9 A resolution from a diverse set of protein structures. At resolutions higher than 2.3 A the most successful program was ARP/wARP 6.0, which accurately built an average of 90% of the main chain. This system builds somewhat larger fractions of the model than the previous version ARP/wARP 5.1, which accurately built an average of 87% of the main chain. Although not specifically designed for model building into high-resolution maps, MAID and RESOLVE were also quite successful in this resolution regime, typically building approximately 80% of the main chain. At 2.3-2.7 A resolution the MAID and RESOLVE programs automatically built approximately 75% of the main-chain atoms in the protein structures used in these tests, which would significantly accelerate the model-building process. Data sets at lower resolution proved more problematic for these programs, although many of the secondary-structure elements were correctly identified and fitted.
[Show abstract][Hide abstract] ABSTRACT: Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site.
[Show abstract][Hide abstract] ABSTRACT: Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4″-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site.
[Show abstract][Hide abstract] ABSTRACT: The emergence of structure-determination initiatives that employ high-throughput protein crystallography emphasizes the need to establish quality-control methods for screening the resulting models prior to deposition with the public data banks. An in-house database of 26 new protein structures, associated diffraction data and high-quality experimentally determined electron-density maps have been used to develop (i) a set of minimal global quality criteria that a structure must meet before the refinement may be considered completed and (ii) a reliable set of indicators for detecting local errors in protein structures. These criteria have been applied to detecting local errors to a set of structures recently deposited in the Protein Data Bank and it is estimated that about 3% of amino acids are incorrectly modeled.
[Show abstract][Hide abstract] ABSTRACT: This article describes new methods for X-ray crystallographic refinement and nuclear magnetic resonance (NMR) structure determination that are available in the recent release of the X-PLOR software, X-PLOR 98.0. The major new features of the X-PLOR 98.0 software are: (i) the introduction of maximum likelihood methods (Pannu and Read, Acta Crystallogr 1996;A52:659-668) for X-ray crystallographic refinement with structure factor amplitude, intensity and phase probability targets, (ii) the addition of the Andersen thermal coupling method for temperature control during simulated annealing refinements, (iii) a new utility function for converting reflection data in to the X-PLOR format, (iv) validated scripts and performance enhancements for structure determination from NMR distance restraints using torsion angle dynamics, (v) fast code for direct nuclear Oberhauser effect (NOE) refinement using matrix doubling and gaussian quadratures, (vi) methodologies for using ambiguous restraint information to perform automated iterative peak assignment and structure determination (Nilges et al., J Mol Biol 1997;269: 408-422). Additional developments in methodology for refining crystal structures from poor initial models include the implementation of a fast adaptive bulk solvent scattering correction and an energy minimization routine that makes use of second derivative information. Trial crystallographic refinements with an energy minimization protocol that includes these enhancements indicate significantly improved convergence. The quality of the resulting models appears comparable to models obtained from refinement protocols that incorporate torsion angle dynamics. Test applications of the new energy minimizer to NMR structure refinement with using NOE calculations also show improved convergence, leading to more optimized final models.
Proteins Structure Function and Bioinformatics 05/1999; 35(1):25-33. DOI:10.1002/(SICI)1097-0134(19990401)35:13.3.CO;2-M · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The solvent content in protein crystals is typically ∼50% of the total crystal volume. Although most of the solvent is poorly ordered, and is frequently approximated by a structureless electron-density continuum, some solvent molecules are tightly localized by multiple hydrogen bonds to the protein surface and have B factors comparable to the protein atoms. This chapter reviews techniques in X-ray crystallography that may be used for modeling both the disordered and the well-ordered solvent in protein crystals. At higher resolution, the diffraction pattern is dominated by density variations in the protein part of the crystal and the scattering contrast between average protein and solvent densities has little effect. If low-resolution data are included in the refinement and no account is taken of bulk solvent, scattering the data will be badly misfit at low resolution and the overall scale factor between observed and calculated structure factors will be distorted. Two common approximations used to compensate for bulk solvent scattering, one applied in real space and one in reciprocal space, are discussed in the chapter.
Methods in Enzymology 02/1997; 277:344-52. DOI:10.1016/S0076-6879(97)77019-8 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The average contact distance between protein and solvent surface atoms in cubic insulin crystals has been determined from two sets of 15 A resolution neutron diffraction data. A contact distance between the water hydrogen sites and the protein surface that is significantly shorter than the average protein-water oxygen contact distance implies that many water molecules are oriented with hydrogen atoms pointed towards the protein surface. The shape of the protein/solvent interface is consistent with the protein envelope obtained from atomic co-ordinates.
[Show abstract][Hide abstract] ABSTRACT: To determine the distribution of monovalent cations around a protein we have measured anomalous scattering diffraction data from Tl-containing cubic insulin crystals at pH 8 and pH 10.5. The differences between Bijvoet reflection pairs within each set of data were used to calculate anomalous scattering difference maps. Both maps show the same six Tl+ sites, which include two well-ordered Tl+ ions previously identified from isomorphous exchange experiments. The other four sites constitute a second class of cations, which, while much more mobile than the protein atoms, are associated with particular ligating groups. Three of the six Tl+ sites are created exclusively by protein main and side chain carbonyl dipoles rather than negatively charged groups. All of the Tl+ ions are positioned so as to interact with both protein atoms and water molecules. The Tl+ occupancies appear to depend in a complex way on interactions with each other and flexibility in the protein structure. The combined occupancies of these cations are slightly less than is required to neutralize the net protein charge of approximately -2e at pH 8 but account for only about half of the approximately -5e protein charge at pH 10.5. Thus, more disordered counterions, not seen in these Bijvoet anomalous scattering difference maps, are more numerous at higher protein net charge.
Proceedings of the National Academy of Sciences 03/1994; 91(4):1224-8. DOI:10.1073/pnas.91.4.1224 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cubic insulin crystals contain a binding site for monovalent cations in a cavity of the crystal dyad in which the bound cation is ligated by protein atomic dipoles and water molecules. These types of interaction are analogous to interactions that occur in small cation-selective carrier and channel molecules. X-ray diffraction data collected from cubic insulin crystals containing Li+, Na+, K+, NH4+, Rb+, and Tl+ show that (i) the differences in cation size do not cause any large alteration in the protein structure around the cation, and (ii) the bound cation is co-ordinated by one or two water molecules, depending on its ionic radii. The relative binding affinities for cations at this dyad site were obtained from an x-ray diffraction analysis of competition experiments in which crystals were dialyzed in mixtures of Tl+ with Li+, Na+, NH4+, Rb+, or Cs+. These data show that this site provides very little discrimination between Na+, K+, Rb+, and Tl+, some selectivity against the small Li+ and the tetrahedrally shaped NH4+, and stronger selectivity against the larger Cs+. The capacity of this site to bind monovalent cations of different sizes may be accounted for by the small number of protein ligating groups and a change from two ligating waters with Li+ and Na+ to one ligating water with the larger cations.
[Show abstract][Hide abstract] ABSTRACT: In macromolecular crystallography, three-dimensional contour surfaces are useful for interactive computer graphics displays of the protein electron density but are less effective for presenting static images of large volumes of solvent density. A raster-based computer graphics program which displays depth-cued projections of continuous density distributions has been developed to analyze the distribution of solvent atoms in macromolecular crystals. Maps of the water distribution in the cubic insulin crystal show some well-ordered waters, which are bound to surrounding protein atoms by multiple hydrogen bonds, and an ill-defined solvent structure at a greater distance from the protein surface. Molecular dynamics calculations were used to assist in the interpretation of the time-varying solvent structure within two enclosed cavities in the crystal. Two water molecules that ligate a sodium ion were almost immobile during the stimulation but the majority of water molecules were found to move rapidly between the density maxima identified from the crystallographic refinement.
Journal of Molecular Graphics 01/1994; 11(4):218-21, 233. DOI:10.1016/0263-7855(93)80001-8