Molecular modeling of proteins including homology modeling, structure determination, and knowledge-based protein design requires tools to evaluate and refine three-dimensional protein structures. Steric clash is one of the artifacts prevalent in low-resolution structures and homology models. Steric clashes arise due to the unnatural overlap of any two non-bonding atoms in a protein structure. Usually, removal of severe steric clashes in some structures is challenging since many existing refinement programs do not accept structures with severe steric clashes. Here, we present a quantitative approach of identifying steric clashes in proteins by defining clashes based on the Van der Waals repulsion energy of the clashing atoms. We also define a metric for quantitative estimation of the severity of clashes in proteins by performing statistical analysis of clashes in high-resolution protein structures. We describe a rapid, automated and robust protocol, Chiron, which efficiently resolves severe clashes in low-resolution structures and homology models with minimal perturbation in the protein backbone. Benchmark studies highlight the efficiency and robustness of Chiron compared to other widely used methods. We provide Chiron as an automated web server to evaluate and resolve clashes in protein structures that can be further used for more accurate protein design.
Homology modeling; refinement; Chiron; Discrete Molecular Dynamics; Protein Design
Protein structure determination and predictive modeling have long been guided by the paradigm that the peptide backbone has a single, context-independent ideal geometry. Both quantum-mechanics calculations and empirical analyses have shown this is an incorrect simplification in that backbone covalent geometry actually varies systematically as a function of the Φ and Ψ backbone dihedral angles. Here, we use a nonredundant set of ultrahigh-resolution protein structures to define these conformation-dependent variations. The trends have a rational, structural basis that can be explained by avoidance of atomic clashes or optimization of favorable electrostatic interactions. To facilitate adoption of this new paradigm, we have created a conformation-dependent library of covalent bond lengths and bond angles and shown that it has improved accuracy over existing methods without any additional variables to optimize. Protein structures derived both from crystallographic refinement and predictive modeling both stand to benefit from incorporation of the new paradigm.
An important element in homology modeling is the use of rotamers to parameterize the sidechain conformation. Despite the many libraries of sidechain rotamers that have been developed, a number of rotamers have been overlooked, due to the fact that they involve hydrogen atoms.
We identify new, well-populated rotamers that involve the hydroxyl-hydrogen atoms of Ser, Thr and Tyr, and the sulfhydryl-hydrogen atom of Cys, using high-resolution crystal structures (<1.2 Å). Although there were refinement artifacts in these structures, comparison with the electron-density maps allowed the placement of hydrogen atoms involved in hydrogen bonds. The χ2 rotamers in Ser, Thr and Cys are consistent with tetrahedral bonding, while the χ3 rotamers in Tyr are consistent with trigonal-planar bonding. Similar rotamers are found in hydrogen atoms that were computationally placed with the Reduce program from the Richardson lab.
Knowledge of these new rotamers will improve the evaluation of hydrogen-bonding networks in protein structures.
All-atom models derived from moderate-resolution protein crystal structures contain a high frequency of close nonbonded contacts, independent of the major refinement program used for structure determination. All-atom refinement with PrimeX corrects many of these problematic interactions, producing models that are better suited for use in computational chemistry and related applications.
All-atom models are essential for many applications in molecular modeling and computational chemistry. Nonbonded atomic contacts much closer than the sum of the van der Waals radii of the two atoms (clashes) are commonly observed in such models derived from protein crystal structures. A set of 94 recently deposited protein structures in the resolution range 1.5–2.8 Å were analyzed for clashes by the addition of all H atoms to the models followed by optimization and energy minimization of the positions of just these H atoms. The results were compared with the same set of structures after automated all-atom refinement with PrimeX and with nonbonded contacts in protein crystal structures at a resolution equal to or better than 0.9 Å. The additional PrimeX refinement produced structures with reasonable summary geometric statistics and similar R
free values to the original structures. The frequency of clashes at less than 0.8 times the sum of van der Waals radii was reduced over fourfold compared with that found in the original structures, to a level approaching that found in the ultrahigh-resolution structures. Moreover, severe clashes at less than or equal to 0.7 times the sum of atomic radii were reduced 15-fold. All-atom refinement with PrimeX produced improved crystal structure models with respect to nonbonded contacts and yielded changes in structural details that dramatically impacted on the interpretation of some protein–ligand interactions.
H atoms; van der Waals radii; restraints; nonbonded contacts; clashes; molecular geometry; model quality; force fields; refinement; riding H atoms; electrostatics; hydrogen bonds
The prediction of loop structures is considered one of the main challenges in the protein folding problem. Regardless of the dependence of the overall algorithm on the protein data bank, the flexibility of loop regions dictates the need for special attention to their structures. In this article, we present algorithms for loop structure prediction with fixed stem and flexible stem geometry. In the flexible stem geometry problem, only the secondary structure of three stem residues on either side of the loop is known. In the fixed stem geometry problem, the structure of the three stem residues on either side of the loop is also known. Initial loop structures are generated using a probability database for the flexible stem geometry problem, and using torsion angle dynamics for the fixed stem geometry problem. Three rotamer optimization algorithms are introduced to alleviate steric clashes between the generated backbone structures and the side chain rotamers. The structures are optimized by energy minimization using an all atom force field. The optimized structures are clustered using a traveling salesman problem based clustering algorithm. The structures in the densest clusters are then utilized to refine dihedral angle bounds on all amino acids in the loop. The entire procedure is carried out for a number of iterations, leading to improved structure prediction and refined dihedral angle bounds. The algorithms presented in this article has been tested on 3190 loops from the PDBSelect25 data set and on targets from the recently concluded CASP9 community-wide experiment.
Protein Structure Prediction; ASTRO-FOLD; all-atom potential; improved bound generation
Motivation: Quality assessment of protein structures is an important part of experimental structure validation and plays a crucial role in protein structure prediction, where the predicted models may contain substantial errors. Most current scoring functions are primarily designed to rank alternative models of the same sequence supporting model selection, whereas the prediction of the absolute quality of an individual protein model has received little attention in the field. However, reliable absolute quality estimates are crucial to assess the suitability of a model for specific biomedical applications.
Results: In this work, we present a new absolute measure for the quality of protein models, which provides an estimate of the ‘degree of nativeness’ of the structural features observed in a model and describes the likelihood that a given model is of comparable quality to experimental structures. Model quality estimates based on the QMEAN scoring function were normalized with respect to the number of interactions. The resulting scoring function is independent of the size of the protein and may therefore be used to assess both monomers and entire oligomeric assemblies. Model quality scores for individual models are then expressed as ‘Z-scores’ in comparison to scores obtained for high-resolution crystal structures. We demonstrate the ability of the newly introduced QMEAN Z-score to detect experimentally solved protein structures containing significant errors, as well as to evaluate theoretical protein models.
In a comprehensive QMEAN Z-score analysis of all experimental structures in the PDB, membrane proteins accumulate on one side of the score spectrum and thermostable proteins on the other. Proteins from the thermophilic organism Thermatoga maritima received significantly higher QMEAN Z-scores in a pairwise comparison with their homologous mesophilic counterparts, underlining the significance of the QMEAN Z-score as an estimate of protein stability.
Availability: The Z-score calculation has been integrated in the QMEAN server available at: http://swissmodel.expasy.org/qmean.
Supplementary information: Supplementary data are available at Bioinformatics online.
I-TASSER is an automated pipeline for protein tertiary structure prediction using multiple threading alignments and iterative structure assembly simulations. In CASP9 experiments, two new algorithms, QUARK and FG-MD, were added to the I-TASSER pipeline for improving the structural modeling accuracy. QUARK is a de novo structure prediction algorithm used for structure modeling of proteins that lack detectable template structures. For distantly homologous targets, QUARK models are found useful as a reference structure for selecting good threading alignments and guiding the I-TASSER structure assembly simulations. FG-MD is an atomic-level structural refinement program that uses structural fragments collected from the PDB structures to guide molecular dynamics simulation and improve the local structure of predicted model, including hydrogen-bonding networks, torsion angles and steric clashes. Despite considerable progress in both the template-based and template-free structure modeling, significant improvements on protein target classification, domain parsing, model selection, and ab initio folding of beta-proteins are still needed to further improve the I-TASSER pipeline.
protein structure prediction; threading; contact prediction; ab initio folding; CASP
Amino acid substitutions in protein structures often require subtle backbone adjustments that are difficult to model in atomic detail. An improved ability to predict realistic backbone changes in response to engineered mutations would be of great utility for the blossoming field of rational protein design. One model that has recently grown in acceptance is the backrub motion, a low-energy dipeptide rotation with single-peptide counter-rotations, that is coupled to dynamic two-state sidechain rotamer jumps, as evidenced by alternate conformations in very high-resolution crystal structures. It has been speculated that backrubs may facilitate sequence changes equally well as rotamer changes. However, backrub-induced shifts and experimental uncertainty are of similar magnitude for backbone atoms in even high-resolution structures, so comparison of wildtype-vs.-mutant crystal structure pairs is not sufficient to directly link backrubs to mutations. In this study, we use two alternative approaches that bypass this limitation. First, we use a quality-filtered structure database to aggregate many examples for precisely defined motifs with single amino acid differences, and find that the effectively amplified backbone differences closely resemble backrubs. Second, we directly apply a provably-accurate, backrub-enabled protein design algorithm to idealized versions of these motifs, and discover that the lowest-energy computed models match the average-coordinate experimental structures. These results support the hypothesis that backrubs participate in natural protein evolution and validate their continued use for design of synthetic proteins.
Protein design has the potential to generate useful molecules for medicine and chemistry, including sensors, drugs, and catalysts for arbitrary reactions. When protein design is carried out starting from an experimentally determined structure, as is often the case, one important aspect to consider is backbone flexibility, because in response to a mutation the backbone often must shift slightly to reconcile the new sidechain with its environment. In principle, one may model the backbone in many ways, but not all are physically realistic or experimentally validated. Here we study the "backrub" motion, which has been previously documented in atomic detail, but only for sidechain movements within single structures. By a twopronged approach involving both structural bioinformatics and computation with a principled design algorithm, we demonstrate that backrubs are sufficient to explain the backbone differences between mutation-related sets of very precisely defined motifs from the protein structure database. Our findings illustrate that backrubs are useful for describing evolutionary sequence change and, by extension, suggest that they are also appropriate for rational protein design calculations.
For template-based modeling in the CASP8 Critical Assessment of Techniques for Protein Structure Prediction, this work develops and applies six new full-model metrics. They are designed to complement and add value to the traditional template-based assessment by GDT (Global Distance Test) and related scores (based on multiple superpositions of Cα atoms between target structure and predictions labeled “model 1”). The new metrics evaluate each predictor group on each target, using all atoms of their best model with above-average GDT. Two metrics evaluate how “protein-like” the predicted model is: the MolProbity score used for validating experimental structures, and a mainchain reality score using all-atom steric clashes, bond length and angle outliers, and backbone dihedrals. Four other new metrics evaluate match of model to target for mainchain and sidechain hydrogen bonds, sidechain end positioning, and sidechain rotamers. Group-average Z-score across the six full-model measures is averaged with group-average GDT Z-score to produce the overall ranking for full-model, high-accuracy performance.
Separate assessments are reported for specific aspects of predictor-group performance, such as robustness of approximately correct template or fold identification, and self-scoring ability at identifying the best of their models. Fold identification is distinct from but correlated with group-average GDT Z-score if target difficulty is taken into account, while self-scoring is done best by servers and is uncorrelated with GDT performance. Outstanding individual models on specific targets are identified and discussed. Predictor groups excelled at different aspects, highlighting the diversity of current methodologies. However, good full-model scores correlate robustly with high Cα accuracy.
homology modeling; protein structure prediction; all-atom contacts; full-model assessment
A wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures.
The construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions.
This study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in the protein core and/or a better packing of alpha-helices with the rest of the structure, caused by the presence of hydrophobic beta-branched side chains.
The protein databank now contains the structures of over 11,000 ligands bound to proteins. These structures are invaluable in applied areas such as structure-based drug design, but are also the substrate for understanding the energetics of intermolecular interactions with proteins. Despite their obvious importance, the careful analysis of ligands bound to protein structures lags behind the analysis of the protein structures themselves. We present an analysis of the geometry of ligands bound to proteins and highlight the role of small molecule crystal structures in enabling molecular modellers to critically evaluate a ligand model’s quality and investigate protein-induced strain.
Electronic supplementary material
The online version of this article (doi:10.1007/s10822-011-9538-6) contains supplementary material, which is available to authorized users.
X-ray refinement; Structure validation; Ligand strain; CSD; PDB; Conformation
In the area of protein structure prediction, recently a lot of effort has gone into the development of Model Quality Assessment Programs (MQAPs). MQAPs distinguish high quality protein structure models from inferior models. Here, we propose a new method to use an MQAP to improve the quality of models. With a given target sequence and template structure, we construct a number of different alignments and corresponding models for the sequence. The quality of these models is scored with an MQAP and used to choose the most promising model. An SVM-based selection scheme is suggested for combining MQAP partial potentials, in order to optimize for improved model selection.
The approach has been tested on a representative set of proteins. The ability of the method to improve models was validated by comparing the MQAP-selected structures to the native structures with the model quality evaluation program TM-score. Using the SVM-based model selection, a significant increase in model quality is obtained (as shown with a Wilcoxon signed rank test yielding p-values below 10-15). The average increase in TMscore is 0.016, the maximum observed increase in TM-score is 0.29.
In template-based protein structure prediction alignment is known to be a bottleneck limiting the overall model quality. Here we show that a combination of systematic alignment variation and modern model scoring functions can significantly improve the quality of alignment-based models.
According to several studies, some nuclear magnetic resonance (NMR) structures are of lower quality, less reliable and less suitable for structural analysis than high-resolution X-ray crystallographic structures. We present a public database of 2405 refined NMR solution structures [statistical torsion angle potentials (STAP) refinement of the NMR database, http://psb.kobic.re.kr/STAP/refinement] from the Protein Data Bank (PDB). A simulated annealing protocol was employed to obtain refined structures with target potentials, including the newly developed STAP. The refined database was extensively analysed using various quality indicators from several assessment programs to determine the nuclear Overhauser effect (NOE) completeness, Ramachandran appearance, χ1-χ2 rotamer normality, various parameters for protein stability and other indicators. Most quality indicators are improved in our protocol mainly due to the inclusion of the newly developed knowledge-based potentials. This database can be used by the NMR structure community for further development of research and validation tools, structure-related studies and modelling in many fields of research.
Nitrosation of protein sulfhydryl groups to form thionitrites (S-nitrosothiols) has been reported to be important in the biochemistry of nitric oxide. Such S-nitrosation of protein thiol residues has been shown to alter the function of some proteins. In this brief communication, we report the X-ray crystal structure of S-nitroso-l-cysteine ethyl ester hydrochloride. Two rotamers with respect to the N—C—C—S moiety are present in the crystal: the major rotamer is in the gauche+ conformation, and the minor rotamer is in the rare anti (trans, antiperiplanar) conformation for a cysteinyl compound. Importantly, the C—S—N=O groups for both rotamers are in the syn (cis, synperiplanar) form. To the best of our knowledge, this is the first reported high-resolution solid-state structure of an S-nitroso derivative of a cysteine or cysteinyl-containing compound.
Nitrosothiol; Thionitrite; Nitrosation; Cysteine; X-ray
Motivation: Programs that evaluate the quality of a protein structural model are important both for validating the structure determination procedure and for guiding the model-building process. Such programs are based on properties of native structures that are generally not expected for faulty models. One such property, which is rarely used for automatic structure quality assessment, is the tendency for conserved residues to be located at the structural core and for variable residues to be located at the surface.
Results: We present ConQuass, a novel quality assessment program based on the consistency between the model structure and the protein's conservation pattern. We show that it can identify problematic structural models, and that the scores it assigns to the server models in CASP8 correlate with the similarity of the models to the native structure. We also show that when the conservation information is reliable, the method's performance is comparable and complementary to that of the other single-structure quality assessment methods that participated in CASP8 and that do not use additional structural information from homologs.
Availability: A perl implementation of the method, as well as the various perl and R scripts used for the analysis are available at http://bental.tau.ac.il/ConQuass/.
Supplementary information: Supplementary data are available at Bioinformatics online.
The RosettaAntibody server (http://antibody.graylab.jhu.edu) predicts the structure of an antibody variable region given the amino-acid sequences of the respective light and heavy chains. In an initial stage, the server identifies and displays the most sequence homologous template structures for the light and heavy framework regions and each of the complementarity determining region (CDR) loops. Subsequently, the most homologous templates are assembled into a side-chain optimized crude model, and the server returns a picture and coordinate file. For users requesting a high-resolution model, the server executes the full RosettaAntibody protocol which additionally models the hyper-variable CDR H3 loop. The high-resolution protocol also relieves steric clashes by optimizing the CDR backbone torsion angles and by simultaneously perturbing the relative orientation of the light and heavy chains. RosettaAntibody generates 2000 independent structures, and the server returns pictures, coordinate files, and detailed scoring information for the 10 top-scoring models. The 10 models enable users to use rational judgment in choosing the best model or to use the set as an ensemble for further studies such as docking. The high-resolution models generated by RosettaAntibody have been used for the successful prediction of antibody–antigen complex structures.
The automation of protein structure determination is an essential component for high-throughput structural analysis in protein X-ray crystallography and is a key element in structural genomics. This highly challenging undertaking relies at present on the availability of high-quality native and derivatized protein crystals diffracting to high or moderate resolution, respectively. Obtaining such crystals often requires significant effort. The present study demonstrates that phases obtained at low resolution (>3.0 Å) from crystals of SeMet-labeled protein can be successfully used for automated structure determination. The crystal structure of acetate CoA-transferase α-subunit was solved using 3.4 Å multiwavelength anomalous dispersion data collected from a crystal containing SeMet-substituted protein and 1.9 Å data collected from a native protein crystal.
X-ray analysis of anti-HIV actinohivin in complex with the target α(1-2)mannobiose moiety of high-mannose type glycans attached to HIV-1 gp120 reveals that the three rotamers generated with 120 rotations around the molecular pseudo-rotation axis are packed randomly in the unit cell according to the P212121 symmetry to exhibit an apparent space group P213 as the statistical structure. However, the high-resolution X-ray structure shows the detailed interaction geometry for specific binding.
Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the α(1–2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C—H⋯O hydrogen bonds facilitated by the α(1–2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O3 and O4 atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O1 atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120.
anti-HIV lectins; actinohivin; high-mannose-type glycan
Rotamer libraries are a valuable tool for protein structure determination, modeling and design. Site-directed tryptophan fluorescence (SDTF) was used in combination with the rotamer model for the fluorescence intensity decays to solve α-helical conformations of proteins in solution. Single Trp mutations located in an α-helical segment of human tear lipocalin were explored for structure assignment. Along with fluorescence λmax values, the rotamer model assignment of fluorescence lifetimes fits the backbone conformation. Typically Trp fluorescence in proteins shows three lifetimes. However, for the α-helix, two lifetimes assigned to t and g− rotamers were satisfactory to describe Trp fluorescence intensity decays. The g+ rotamer is not feasible in the α-helix due to steric restriction. Trp rotamer distributions obtained by fluorescence were compared with the rotamer library derived from X-ray crystallography data of proteins. The Trp rotamer distributions vary for solvent exposed and buried (tertiary interaction) sites. A new strategy using the rotamer distribution with SDTF (RD-SDTF) removes the limitation of regular SDTF and other labeling techniques, in which site specific differences, e.g. accessibility, are presumed. The RD-SDTF technique does not rely on environmental differences of side chains and is able to detect α-helical structure where all side chains are exposed to solvent. Potentially this technique is applicable to various proteins including membrane proteins, which are rich in α-helix motif.
LCN1; fluorescence lifetime; side chain conformations; protein structure
Using the harmonic-approximation approach of the accompanying paper and AM1 energy surfaces of terminally-blocked amino-acid residues, we determined physics-based side-chain-rotamer potentials and the side-chain virtual-bond-deformation potentials of 19 natural amino-acid residues with side chains. The potentials were approximated by analytical formulas and implemented in the UNRES mesoscopic dynamics program. For comparison, the corresponding statistical potentials were determined from 19,682 high-resolution protein structures. The low-free-energy region of both the AM1-derived and the statistical potentials is determined by the valence geometry and the L-chirality, and its size increases with side-chain flexibility and decreases with increasing virtual-bond-angle θ. The differences between the free energies of rotamers are greater for the AM1-derived potentials compared to the statistical potentials and, for alanine and other residues with small side chains, a region corresponding to the Cax7 conformation has remarkably low free energy for the AM1-derived potentials, as opposed to the statistical potentials. These differences probably result from the interactions between neighboring residues and indicate the need for introduction of cooperative terms accounting for the coupling between side-chain-rotamer and backbone interactions. Both AM1-derived and statistical virtual-bond-deformation potentials are multimodal for flexible side chains and are topologically similar; however, the regions of minima of the statistical potentials are much narrower, which probably results from imposing restraints in structure determination. The force field with the new potentials was preliminarily optimized using the FBP WW domain (1E0L) and the engrailed homeodomain (1ENH) as training proteins and assessed to be reasonably transferable.
protein folding; UNRES force field; local-interaction potentials; molecular quantum mechanics; harmonic approximation
Motivation: The assessment of protein structure prediction techniques requires objective criteria to measure the similarity between a computational model and the experimentally determined reference structure. Conventional similarity measures based on a global superposition of carbon α atoms are strongly influenced by domain motions and do not assess the accuracy of local atomic details in the model.
Results: The Local Distance Difference Test (lDDT) is a superposition-free score that evaluates local distance differences of all atoms in a model, including validation of stereochemical plausibility. The reference can be a single structure, or an ensemble of equivalent structures. We demonstrate that lDDT is well suited to assess local model quality, even in the presence of domain movements, while maintaining good correlation with global measures. These properties make lDDT a robust tool for the automated assessment of structure prediction servers without manual intervention.
Availability and implementation: Source code, binaries for Linux and MacOSX, and an interactive web server are available at http://swissmodel.expasy.org/lddt
Supplementary information: Supplementary data are available at Bioinformatics online.
The crystal structure of the ternary complex of NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. C-1 with the cofactor (NAD+) and the inhibitor (azide ion) was established at 1.1 A resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacitalicents parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the titalicperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 A resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreitalicent with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction.
The NMSim web server implements a three-step approach for multiscale modeling of protein conformational changes. First, the protein structure is coarse-grained using the FIRST software. Second, a rigid cluster normal-mode analysis provides low-frequency normal modes. Third, these modes are used to extend the recently introduced idea of constrained geometric simulations by biasing backbone motions of the protein, whereas side chain motions are biased toward favorable rotamer states (NMSim). The generated structures are iteratively corrected regarding steric clashes and stereochemical constraint violations. The approach allows performing three simulation types: unbiased exploration of conformational space; pathway generation by a targeted simulation; and radius of gyration-guided simulation. On a data set of proteins with experimentally observed conformational changes, the NMSim approach has been shown to be a computationally efficient alternative to molecular dynamics simulations for conformational sampling of proteins. The generated conformations and pathways of conformational transitions can serve as input to docking approaches or more sophisticated sampling techniques. The web server output is a trajectory of generated conformations, Jmol representations of the coarse-graining and a subset of the trajectory and data plots of structural analyses. The NMSim webserver, accessible at http://www.nmsim.de, is free and open to all users with no login requirement.
Motivation: Macromolecular crystal structures in the Protein Data Bank (PDB) are a key source of structural insight into biological processes. These structures, some >30 years old, were constructed with methods of their era. With PDB_REDO, we aim to automatically optimize these structures to better fit their corresponding experimental data, passing the benefits of new methods in crystallography on to a wide base of non-crystallographer structure users.
Results: We developed new algorithms to allow automatic rebuilding and remodeling of main chain peptide bonds and side chains in crystallographic electron density maps, and incorporated these and further enhancements in the PDB_REDO procedure. Applying the updated PDB_REDO to the oldest, but also to some of the newest models in the PDB, corrects existing modeling errors and brings these models to a higher quality, as judged by standard validation methods.
Availability and Implementation: The PDB_REDO database and links to all software are available at http://www.cmbi.ru.nl/pdb_redo.
Contact: email@example.com; firstname.lastname@example.org
Supplementary Information: Supplementary data are available at Bioinformatics online.
PROSESS (PROtein Structure Evaluation Suite and Server) is a web server designed to evaluate and validate protein structures generated by X-ray crystallography, NMR spectroscopy or computational modeling. While many structure evaluation packages have been developed over the past 20 years, PROSESS is unique in its comprehensiveness, its capacity to evaluate X-ray, NMR and predicted structures as well as its ability to evaluate a variety of experimental NMR data. PROSESS integrates a variety of previously developed, well-known and thoroughly tested methods to evaluate both global and residue specific: (i) covalent and geometric quality; (ii) non-bonded/packing quality; (iii) torsion angle quality; (iv) chemical shift quality and (v) NOE quality. In particular, PROSESS uses VADAR for coordinate, packing, H-bond, secondary structure and geometric analysis, GeNMR for calculating folding, threading and solvent energetics, ShiftX for calculating chemical shift correlations, RCI for correlating structure mobility to chemical shift and PREDITOR for calculating torsion angle-chemical shifts agreement. PROSESS also incorporates several other programs including MolProbity to assess atomic clashes, Xplor-NIH to identify and quantify NOE restraint violations and NAMD to assess structure energetics. PROSESS produces detailed tables, explanations, structural images and graphs that summarize the results and compare them to values observed in high-quality or high-resolution protein structures. Using a simplified red–amber–green coloring scheme PROSESS also alerts users about both general and residue-specific structural problems. PROSESS is intended to serve as a tool that can be used by structure biologists as well as database curators to assess and validate newly determined protein structures. PROSESS is freely available at http://www.prosess.ca.