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Protein Crystal Structures (protein + crystal_structure)
Selected AbstractsTracking Flavin Conformations in Protein Crystal Structures with Raman Spectroscopy and QM/MM Calculations,ANGEWANDTE CHEMIE, Issue 13 2010Kjendseth Røhr, Åsmund Strahlenschäden: Für eine aussagekräftige Analyse von Flavoenzym-Kristallstrukturen muss die Cofaktorkonformation genau bekannt sein. Doch durch Röntgenstrahlen während der Datensammlung erzeugte Photoelektronen können den Flavincofaktor reduzieren und so seine Geometrie ändern (siehe Bild). Parallel zur Datensammlung beobachtete Raman-Schwingungsmoden lieferten wichtige Informationen über den tatsächlichen Flavinzustand. [source] Validation of crystallographic models containing TLS or other descriptions of anisotropyACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Frank Zucker The use of TLS (translation/libration/screw) models to describe anisotropic displacement of atoms within a protein crystal structure has become increasingly common. These models may be used purely as an improved methodology for crystallographic refinement or as the basis for analyzing inter-domain and other large-scale motions implied by the crystal structure. In either case it is desirable to validate that the crystallographic model, including the TLS description of anisotropy, conforms to our best understanding of protein structures and their modes of flexibility. A set of validation tests has been implemented that can be integrated into ongoing crystallographic refinement or run afterwards to evaluate a previously refined structure. In either case validation can serve to increase confidence that the model is correct, to highlight aspects of the model that may be improved or to strengthen the evidence supporting specific modes of flexibility inferred from the refined TLS model. Automated validation checks have been added to the PARVATI and TLSMD web servers and incorporated into the CCP4i user interface. [source] Escherichia coli MltA: MAD phasing and refinement of a tetartohedrally twinned protein crystal structureACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2005Thomas R. M. Barends Crystals were grown of a mutant form of the bacterial cell-wall maintenance protein MltA that diffracted to 2.15,Å resolution. When phasing with molecular replacement using the native structure failed, selenium MAD was used to obtain initial phases. However, after MAD phasing the crystals were found to be tetartohedrally twinned, hampering correct space-group determination and refinement. A refinement protocol was designed to take tetartohedral twinning into account and was successfully applied to refine the structure. The refinement protocol is described and the reasons for the failure of molecular replacement and the success of MAD are discussed in terms of the effects of the tetartohedral twinning. [source] Removing bias from solvent atoms in electron density mapsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2008Eric N. Brown Atomic structures of proteins determined via protein crystallography contain numerous solvent atoms. The experimental data for the determination of a water molecule's O-atom position is often a small contained blob of unidentified electron density. Unfortunately, the nature of crystallographic refinement lets poorly placed solvent atoms bias the future refined positions of all atoms in the crystal structure. This research article presents the technique of omit-maps applied to remove the bias introduced by poorly determined solvent atoms, enabling the identification of incorrectly placed water molecules in partially refined crystal structures. A total of 160 protein crystal structures with 45,912 distinct water molecules were processed using this technique. Most of the water molecules in the deposited structures were well justified. However, a few of the solvent atoms in this test data set changed appreciably in position, displacement parameter or electron density when fitted to the solvent omit-map, raising questions about how much experimental support exists for these solvent atoms. [source] About the efficiency of the early FOMs in ab initio protein phasingJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2004Maria C. Burla All ab initio techniques for solving protein crystal structures use multisolution approaches. Several figures of merit that are found in the literature are efficient in the last steps of the phasing process, when some trials converge to the correct solution with a relatively small average phase error. Early figures of merit are much more critical; they should be able to recognize useful trials when the phase error is still large, and their efficiency determines the efficiency of the program. In the present work, a wide variety of known figures of merit at atomic and quasi-atomic (,1.4,Å) resolution have been tested; new figures have also been devised and tested. Their application to a large set of test structures allows the study of their properties at different data resolutions and the selection of the most efficient figures within the SIR2003-N framework. [source] XAFS studies of nitrogenase: the MoFe and VFe proteins and the use of crystallographic coordinates in three-dimensional EXAFS data analysisJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2003Richard W. Strange This paper reports a three-dimensional EXAFS refinement of the Mo coordination sphere of the FeMoco cluster of the dithionite-reduced MoFe protein from Klebsiella pneumoniae nitrogenase (Kp1) using the 1.6,Å-resolution crystallographic coordinates. At this resolution, the positions of the heavy (Fe and S) atoms of the cluster are well determined and there is excellent agreement between the crystallographic and EXAFS models. However, the lighter homocitrate and histidine ligands are poorly determined in the crystal structure, and it is shown that the application of EXAFS-derived distance restraints during the early stages of crystallographic refinement provides a means of substantially improving (by ,0.1,Å) the final crystallographic model. The consistency of the EXAFS analysis with the crystallographic information in this case justifies applications of EXAFS to cases where protein crystal structures are absent. Thus, the VFe protein of V-nitrogenase has been shown by EXAFS to possess a V-atom site catalytically similar to the well characterized MoFe-nitrogenases, with V replacing Mo. [source] Structures of the OmpF porin crystallized in the presence of foscholine-12PROTEIN SCIENCE, Issue 5 2010Georgia Kefala Abstract The endogenous Escherichia coli porin OmpF was crystallized as an accidental by-product of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine-12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a = 136.7 Å, b = 210.5 Å, c = 137 Å, and , = 100.5°, and the resolution of 3.8 Å. The second structure was solved at the resolution of 4.4 Å and was refined in the P321 space group, with unit cell parameters a = 215.5 Å, b = 215.5 Å, c = 137.5 Å, and , = 120°. Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli. [source] Mining protein dynamics from sets of crystal structures using "consensus structures"PROTEIN SCIENCE, Issue 4 2010Gerard J. P. van Westen Abstract In this work, we describe two novel approaches to utilize the dynamic structure information implicitly contained in large crystal structure data sets. The first approach visualizes both consistent as well as variable ligand-induced changes in ligand-bound compared with apo protein crystal structures. For this purpose, information was mined from B-factors and ligand-induced residue displacements in multiple crystal structures, minimizing experimental error and noise. With this approach, the mechanism of action of non-nucleoside reverse transcriptase inhibitors (NNRTIs) as an inseparable combination of distortion of protein dynamics and conformational changes of HIV-1 reverse transcriptase was corroborated (a combination of the previously proposed "molecular arthritis" and "distorted site" mechanisms). The second approach presented here uses "consensus structures" to map common binding features that are present in a set of structures of NNRTI-bound HIV-1 reverse transcriptase. Consensus structures are based on different levels of structural overlap of multiple crystal structures and are used to analyze protein,ligand interactions. The structures are shown to yield information about conserved hydrogen bonding interactions as well as binding-pocket flexibility, shape, and volume. From the consensus structures, a common wild type NNRTI binding pocket emerges. Furthermore, we were able to identify a conserved backbone hydrogen bond acceptor at P236 and a novel hydrophobic subpocket, which are not yet utilized by current drugs. Our methods introduced here reinterpret the atom information and make use of the data variability by using multiple structures, complementing classical 3D structural information of single structures. [source] Determination of ,-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theoryPROTEIN SCIENCE, Issue 4 2000Jia Ke Sun Abstract Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the ,-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol,1 compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol,1 less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1. [source] The statistics of the highest E valueACTA CRYSTALLOGRAPHICA SECTION A, Issue 4 2007Grzegorz Chojnowski In a previous publication, the Gumbel,Fisher,Tippett (GFT) extreme-value analysis has been applied to investigate the statistics of the intensity of the strongest reflection in a thin resolution shell. Here, a similar approach is applied to study the distribution, expectation value and standard deviation of the highest normalized structure-factor amplitude (E value). As before, acentric and centric reflections are treated separately, a random arrangement of scattering atoms is assumed, and E -value correlations are neglected. Under these assumptions, it is deduced that the highest E value is GFT distributed to a good approximation. Moreover, it is shown that the root of the expectation value of the highest `normalized' intensity is not only an upper limit for the expectation value of the highest E value but also a very good estimate. Qualitatively, this can be attributed to the sharpness of the distribution of the highest E value. Although the formulas were derived with various simplifying assumptions and approximations, they turn out to be useful also for real small-molecule and protein crystal structures, for both thin and thick resolution shells. The only limitation is that low-resolution data (below 2.5,Å) have to be excluded from the analysis. These results have implications for the identification of outliers in experimental diffraction data. [source] On the application of an experimental multipolar pseudo-atom library for accurate refinement of small-molecule and protein crystal structuresACTA CRYSTALLOGRAPHICA SECTION A, Issue 2 2007Bartosz Zarychta With an increasing number of biomacromolecular crystal structures being measured to ultra-high resolution, it has become possible to extend to large systems experimental charge-density methods that are usually applied to small molecules. A library has been built of average multipole populations describing the electron density of chemical groups in all 20 amino acids found in proteins. The library uses the Hansen & Coppens multipolar pseudo-atom model to derive molecular electron density and electrostatic potential distributions. The library values are obtained from several small peptide or amino acid crystal structures refined against ultra-high-resolution X-ray diffraction data. The library transfer is applied automatically in the MoPro software suite to peptide and protein structures measured at atomic resolution. The transferred multipolar parameters are kept fixed while the positional and thermal parameters are refined. This enables a proper deconvolution of thermal motion and valence-electron-density redistributions, even when the diffraction data do not extend to subatomic resolution. The use of the experimental library multipolar atom model (ELMAM) also has a major impact on crystallographic structure modelling in the case of small-molecule crystals at atomic resolution. Compared to a spherical-atom model, the library transfer results in a more accurate crystal structure, notably in terms of thermal displacement parameters and bond distances involving H atoms. Upon transfer, crystallographic statistics of fit are improved, particularly free R factors, and residual electron-density maps are cleaner. [source] An experimental charge density of HEPESACTA CRYSTALLOGRAPHICA SECTION B, Issue 4 2010We report the experimental charge density of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], which is a common buffering agent. The structure was refined using the Hansen,Coppens formalism. The ability of the HEPES molecule to form stable intermolecular interactions and intermolecular hydrogen bonds in the crystal structure is discussed in terms of its buffering properties. The protonation mode observed in the crystal structure is different from that expected in solution, suggesting that additional factors must be taken into consideration in order to explain the solution properties of the compound. As ordered HEPES molecules are found in the active sites of proteins in several protein crystal structures, our results will allow for quantitative analysis of the electrostatic potential of the interacting surfaces of those proteins. [source] Getting the best out of long-wavelength X-rays: de novo chlorine/sulfur SAD phasing of a structural protein from ATVACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2010Adeline Goulet The structure of a 14,kDa structural protein from Acidianus two-tailed virus (ATV) was solved by single-wavelength anomalous diffraction (SAD) phasing using X-ray data collected at 2.0,Å wavelength. Although the anomalous signal from methionine sulfurs was expected to suffice to solve the structure, one chloride ion turned out to be essential to achieve phasing. The minimal data requirements and the relative contributions of the Cl and S atoms to phasing are discussed. This work supports the feasibility of a systematic approach for the solution of protein crystal structures by SAD based on intrinsic protein light atoms along with associated chloride ions from the solvent. In such cases, data collection at long wavelengths may be a time-efficient alternative to selenomethionine substitution and heavy-atom derivatization. [source] Application of maximum-entropy maps in the accurate refinement of a putative acylphosphatase using 1.3,Å X-ray diffraction dataACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2008Eiji Nishibori Accurate structural refinement of a putative acylphosphatase using 1.3,Å X-ray diffraction data was carried out using charge densities determined by the maximum-entropy method (MEM). The MEM charge density clearly revealed detailed features of the solvent region of the putative acylphosphatase crystalline structure, some of which had never been observed in conventional Fourier maps. The structural model in the solvent region was constructed as distributions of anisotropic water atoms. The omit-difference MEM maps and the difference MEM maps were effective in revealing details of the protein structure, such as multiple conformations of the side chains of amino-acid residues, anisotropy of atoms and H atoms. By model building using the MEM charge densities, the reliability factors R1 and Rfree in the SHELX refinement were dramatically improved from 17.9% and 18.3% to 9.6% and 10.0%, respectively. The present results prove the usefulness of MEM in improving the accuracy of refinement of protein crystal structures. [source] Quality of protein crystal structuresACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2007Eric N. Brown The genomics era has seen the propagation of numerous databases containing easily accessible data that are routinely used by investigators to interpret results and generate new ideas. Most investigators consider data extracted from scientific databases to be error-free. However, data generated by all experimental techniques contain errors and some, including the coordinates in the Protein Data Bank (PDB), also integrate the subjective interpretations of experimentalists. This paper explores the determinants of protein structure quality metrics used routinely by protein crystallographers. These metrics are available for most structures in the database, including the R factor, Rfree, real-space correlation coefficient, Ramachandran violations etc. All structures in the PDB were analyzed for their overall quality based on nine different quality metrics. Multivariate statistical analysis revealed that while technological improvements have increased the number of structures determined, the overall quality of structures has remained constant. The quality of structures deposited by structural genomics initiatives are generally better than the quality of structures from individual investigator laboratories. The most striking result is the association between structure quality and the journal in which the structure was first published. The worst offenders are the apparently high-impact general science journals. The rush to publish high-impact work in the competitive atmosphere may have led to the proliferation of poor-quality structures. [source] Refinement of protein crystal structures using energy restraints derived from linear-scaling quantum mechanicsACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2005Ning Yu A novel method is proposed in which combined restraints derived from linear-scaling semiempirical quantum-mechanical (QM) calculations and X-ray diffraction data are combined to refine crystal structures of proteins. Its performance has been tested on a small protein molecule, bovine pancreatic trypsin inhibitor (BPTI). The refinement involves minimization of the sum of a geometric energy function and an X-ray target function based on either the least-squares residual or the maximum-likelihood formalism. For comparison, similar refinement runs have also been performed using energy restraints derived from the force field available in the Crystallography & NMR System (CNS) program. The QM refinements were carried out with weights that were varied by several orders of magnitude and the optimal weights were identified by observing the trend in the final free R values, QM heats of formation and coordinate root-mean-square deviations (r.m.s.d.s) from the crystal structure. It is found that the QM weights are typically smaller but generally on the same scale as the molecular-mechanics (MM) weights for the respective X-ray target functions. The crystallographic R, free R, real-space R values and correlation coefficients based on the structures refined with the energy restraints derived from our QM calculations and Engh and Huber parameters are comparable, suggesting that the QM restraints are capable of maintaining reasonable stereochemistry to a similar degree as the force-field parameters. A detailed inspection of the structures refined with the QM and MM energy restraints reveals that one of the common differences between them and the crystal structure is that the strained bond angles in the crystal structure are corrected after energetically restrained refinements. Systematic differences in certain bond lengths between the QM-refined structures and the statistical averages of experimental structures have also been observed and discussed. [source] Validation of protein crystal structuresACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2000Gerard J. Kleywegt Since the process of building and refining a model of a biomacromolecule based on crystallographic data is subjective, quality-control techniques are required to assess the validity of such models. During the 1990s, much experience was gained; the methods used and some of the lessons learned are reviewed here. In addition, an extensive compendium of quality criteria and quality-control methods that are or have been used to validate models of biomacromolecules has been compiled. The emphasis in this compendium is on the validation of protein crystal structures. [source] Use of thallium to identify monovalent cation binding sites in GroELACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Philip D. Kiser GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D7 symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg2+ and K+, to bind and hydrolyze ATP. A K+ -binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K+ -binding site may exist within GroEL. Because K+ has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K+ mimetic Tl+ was incorporated into GroEL crystals, a moderately redundant 3.94,Å resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K+ next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl+ for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low. [source] | ||||||||||