Evaluating protein structures determined by structural genomics consortia

Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X-ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely-used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283-291), MolProbity (Lovell et al., Proteins 2003;50:437-450), Verify3D (Luthy et al., Nature 1992;356:83-85), ProsaII (Sippl, Proteins 1993;17: 355-362), the PDB validation software, and various structure-validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness-of-fit between structures and experimental data, and knowledge-based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site-specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5-year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher-resolution X-ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this "structure quality score gap" between NMR and X-ray crystal structures are discussed.     

Assessing model accuracy using the homology modeling automatically software

Homology modeling is a powerful technique that greatly increases the value of experimental structure determination by using the structural information of one protein to predict the structures of homologous proteins. We have previously described a method of homology modeling by satisfaction of spatial restraints (Li et al., Protein Sci 1997;6:956-970). The Homology Modeling Automatically (HOMA) web site, , is a new tool, using this method to predict 3D structure of a target protein based on the sequence alignment of the target protein to a template protein and the structure coordinates of the template. The user is presented with the resulting models, together with an extensive structure validation report providing critical assessments of the quality of the resulting homology models. The homology modeling method employed by HOMA was assessed and validated using twenty-four groups of homologous proteins. Using HOMA, homology models were generated for 510 proteins, including 264 proteins modeled with correct folds and 246 modeled with incorrect folds. Accuracies of these models were assessed by superimposition on the corresponding experimentally determined structures. A subset of these results was compared with parallel studies of modeling accuracy using several other automated homology modeling approaches. Overall, HOMA provides prediction accuracies similar to other state-of-the-art homology modeling methods. We also provide an evaluation of several structure quality validation tools in assessing the accuracy of homology models generated with HOMA. This study demonstrates that Verify3D (Luthy et al., Nature 1992;356:83-85) and ProsaII (Sippl, Proteins 1993;17:355-362) are most sensitive in distinguishing between homology models with correct or incorrect folds. For homology models that have the correct fold, the steric conformational energy (including primarily the Van der Waals energy), MolProbity clashscore (Word et al., Protein Sci 2000;9:2251-2259), and the PROCHECK G-factors (Laskowski et al., J Biomol NMR 1996;8:477-486) provide sensitive and consistent methods for assessing accuracy and can distinguish between homology models of higher and lower accuracy. As demonstrated in the accompanying paper (Bhattacharya et al., accompanying paper), combinations of these scores for models generated with HOMA provide a basis for distinguishing low from high accuracy models.     

Building proteins from C alpha coordinates using the dihedral probability grid Monte Carlo method.

Dihedral probability grid Monte Carlo (DPG-MC) is a general-purpose method of conformational sampling that can be applied to many problems in peptide and protein modeling. Here we present the DPG-MC method and apply it to predicting complete protein structures from C alpha coordinates. This is useful in such endeavors as homology modeling, protein structure prediction from lattice simulations, or fitting protein structures to X-ray crystallographic data. It also serves as an example of how DPG-MC can be applied to systems with geometric constraints. The conformational propensities for individual residues are used to guide conformational searches as the protein is built from the amino-terminus to the carboxyl-terminus. Results for a number of proteins show that both the backbone and side chain can be accurately modeled using DPG-MC. Backbone atoms are generally predicted with RMS errors of about 0.5 A (compared to X-ray crystal structure coordinates) and all atoms are predicted to an RMS error of 1.7 A or better.     

Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.     

Molecular dynamics model structures for the molten globule state of alpha-lactalbumin: aromatic residue clusters I and II

To model the molten globule structure of alpha-lactalbumin, molecular dynamics (MD) simulations were carried out for the protein in explicit water at high temperature. In these simulations, long-range Coulomb interactions were evaluated explicitly with an original method (particle-particle and particle-cell: PPPC) to avoid artifacts caused by the cut-off. The MD simulations were started from two initial conditions to verify that similar results would be obtained. From the last 150 ps trajectories of the two MD simulations, two partially unfolded average structures were obtained. These structures had the following common structural features which are characteristic of the molten globule state. The radii of gyration for these conformations were 7.4 and 9.6% larger than that of the native state. These values were almost the same as the experimental value (9.6%) observed recently by small-angle X-ray scattering (Kataoka,M., Kuwajima,K., Tokunaga,F. and Goto,Y., 1997, Protein Sci., 6, 422-430). Furthermore, aromatic residues of clusters I and II in these structures were far apart from each other except for Try103-Trp104. This result is in good agreement with NMR experimental results for the acid-denatured molten globule state (Alexandrescu et al., 1992, 1993); that is, NOE signals between the aromatic residues were not observed, except for that of Try103-Trp104 in the molten globule state. Other structural features of these models for the molten globule state are discussed with reference to native state structures.     

Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy

The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].     

Molecular modeling study of the editing active site of Escherichia coli leucyl-tRNA synthetase: two amino acid binding sites in the editing domain

Aminoacyl-tRNA synthetases (aaRSs) strictly discriminate their cognate amino acids. Some aaRSs accomplish this via proofreading and editing mechanisms. Mursinna and coworkers recently reported that substituting a highly conserved threonine (T252) with an alanine within the editing domain of Escherichia coli leucyl-tRNA synthetase (LeuRS) caused LeuRS to cleave its cognate aminoacylated leucine from tRNA(Leu) (Mursinna et al., Biochemistry 2001;40:5376-5381). To achieve atomic level insight into the role of T252 in LeuRS and the editing reaction of aaRSs, a series of molecular modeling studies including homology modeling and automated docking simulations were carried out. A 3D structure of E. coli LeuRS was constructed via homology modeling using the X-ray structure of Thermus thermophilus LeuRS as a template because the E. coli LeuRS structure is not available from X-ray or NMR studies. However, both the X-ray T. thermophilus and homology-modeled E. coli structures were used in our studies. Amino acid binding sites in the proposed editing domain, which is also called the connective polypeptide 1 (CP1) domain, were investigated by automated docking studies. The root mean square deviation (RMSD) for backbone atoms between the X-ray and homology-modeled structures was 1.18 A overall and 0.60 A for the editing (CP1) domain. Automated docking studies of a leucine ligand into the editing domain were performed for both structures: homology structure of E. coli LeuRS and X-ray structure of T. thermophilus LeuRS for comparison. The results of the docking studies suggested that there are two possible amino acid binding sites in the CP1 domain for both proteins. The first site lies near a threonine-rich region that includes the highly conserved T252 residue, which is important for amino acid discrimination. The second site is located in a flexible loop region surrounded by residues E292, A293, M295, A296, and M298. The important T252 residue is at the bottom of the first binding pocket.     

High-quality homology models derived from NMR and X-ray structures of E. coli proteins YgdK and Suf E suggest that all members of the YgdK/Suf E protein family are enhancers of cysteine desulfurases

The structural biology of proteins mediating iron-sulfur (Fe-S) cluster assembly is central for understanding several important biological processes. Here we present the NMR structure of the 16-kDa protein YgdK from Escherichia coli, which shares 35% sequence identity with the E. coli protein SufE. The SufE X-ray crystal structure was solved in parallel with the YdgK NMR structure in the Northeast Structural Genomics (NESG) consortium. Both proteins are (1) key components for Fe-S metabolism, (2) exhibit the same distinct fold, and (3) belong to a family of at least 70 prokaryotic and eukaryotic sequence homologs. Accurate homology models were calculated for the YgdK/SufE family based on YgdK NMR and SufE crystal structure. Both structural templates contributed equally, exemplifying synergy of NMR and X-ray crystallography. SufE acts as an enhancer of the cysteine desulfurase activity of SufS by SufE-SufS complex formation. A homology model of CsdA, a desulfurase encoded in the same operon as YgdK, was modeled using the X-ray structure of SufS as a template. Protein surface and electrostatic complementarities strongly suggest that YgdK and CsdA likewise form a functional two-component desulfurase complex. Moreover, structural features of YgdK and SufS, which can be linked to their interaction with desulfurases, are conserved in all homology models. It thus appears very likely that all members of the YgdK/SufE family act as enhancers of Suf-S-like desulfurases. The present study exemplifies that "refined" selection of two (or more) targets enables high-quality homology modeling of large protein families.     

Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints.

The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF.     

Non-3D domain swapped crystal structure of truncated zebrafish alphaA crystallin

In previous work on truncated alpha crystallins (Laganowsky et al., Protein Sci 2010; 19:1031–1043), we determined crystal structures of the alpha crystallin core, a seven beta‐stranded immunoglobulin‐like domain, with its conserved C‐terminal extension. These extensions swap into neighboring cores forming oligomeric assemblies. The extension is palindromic in sequence, binding in either of two directions. Here, we report the crystal structure of a truncated alphaA crystallin (AAC) from zebrafish (Danio rerio) revealing C‐terminal extensions in a non three‐dimensional (3D) domain swapped, “closed” state. The extension is quasi‐palindromic, bound within its own zebrafish core domain, lying in the opposite direction to that of bovine AAC, which is bound within an adjacent core domain (Laganowsky et al., Protein Sci 2010; 19:1031–1043). Our findings establish that the C‐terminal extension of alpha crystallin proteins can be either 3D domain swapped or non‐3D domain swapped. This duality provides another molecular mechanism for alpha crystallin proteins to maintain the polydispersity that is crucial for eye lens transparency.     

Atomic-resolution crystal structure of the antiviral lectin scytovirin

The crystal structures of the natural and recombinant antiviral lectin scytovirin (SVN) were solved by single-wavelength anomalous scattering and refined with data extending to 1.3 A and 1.0 A resolution, respectively. A molecule of SVN consists of a single chain 95 amino acids long, with an almost perfect sequence repeat that creates two very similar domains (RMS deviation 0.25 A for 40 pairs of Calpha atoms). The crystal structure differs significantly from a previously published NMR structure of the same protein, with the RMS deviations calculated separately for the N- and C-terminal domains of 5.3 A and 3.7 A, respectively, and a very different relationship between the two domains. In addition, the disulfide bonding pattern of the crystal structures differs from that described in the previously published mass spectrometry and NMR studies.     

Simulated annealing with restrained molecular dynamics using CONGEN: energy refinement of the NMR solution structures of epidermal and type-alpha transforming growth factors.

The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168; Bassolino-Klimas D et al., 1996, Protein Sci 5:593-603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type-alpha transforming growth factor (hTGF alpha). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR-derived constraints for mEGF and 424 NMR-derived constraints for hTGF alpha were used in these energy-refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low-energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., "relaxed") sets of the EGF and hTGF alpha internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy-refined structures of mEGF and hTGF alpha are compared with structures determined previously and with structures of homologous proteins determined by NMR and X-ray crystallography.     

A large data set comparison of protein structures determined by crystallography and NMR: statistical test for structural differences and the effect of crystal packing

The existence of a large number of proteins for which both nuclear magnetic resonance (NMR) and X-ray crystallographic coordinates have been deposited into the Protein Data Bank (PDB) makes the statistical comparison of the corresponding crystal and NMR structural models over a large data set possible, and facilitates the study of the effect of the crystal environment and other factors on structure. We present an approach for detecting statistically significant structural differences between crystal and NMR structural models which is based on structural superposition and the analysis of the distributions of atomic positions relative to a mean structure. We apply this to a set of 148 protein structure pairs (crystal vs NMR), and analyze the results in terms of methodological and physical sources of structural difference. For every one of the 148 structure pairs, the backbone root-mean-square distance (RMSD) over core atoms of the crystal structure to the mean NMR structure is larger than the average RMSD of the members of the NMR ensemble to the mean, with 76% of the structure pairs having an RMSD of the crystal structure to the mean more than a factor of two larger than the average RMSD of the NMR ensemble. On average, the backbone RMSD over core atoms of crystal structure to the mean NMR is approximately 1 A. If non-core atoms are included, this increases to 1.4 A due to the presence of variability in loops and similar regions of the protein. The observed structural differences are only weakly correlated with the age and quality of the structural model and differences in conditions under which the models were determined. We examine steric clashes when a putative crystalline lattice is constructed using a representative NMR structure, and find that repulsive crystal packing plays a minor role in the observed differences between crystal and NMR structures. The observed structural differences likely have a combination of physical and methodological causes. Stabilizing attractive interactions arising from intermolecular crystal contacts which shift the equilibrium of the crystal structure relative to the NMR structure is a likely physical source which can account for some of the observed differences. Methodological sources of apparent structural difference include insufficient sampling or other issues which could give rise to errors in the estimates of the precision and/or accuracy.     

Studies on oligosaccharyl transferase in yeast

In yeast, OT consists of nine different subunits, all of which contain one or more predicted transmembrane segments. In yeast, five of these proteins are encoded by essential genes, Swp1p, Wbp1p, Ost2p, Ost1p and Stt3p. Four others are not essential Ost3p, Ost4p, Ost5p, Ost6p. All yeast OT subunits have been cloned and sequenced (Kelleher et al., 1992; 2003; Kelleher & Gilmore, 1997; Kumar et al., 1994; 1995; Breuer & Bause, 1995) and the structure of one of them, Ost4p, has been solved by NMR (Zubkov et al., 2004). Very recently, the preliminary crystal structure of the lumenal domain of an archaeal Stt3p homolog has been reported (Mayumi et al., 2007). Homologs of all OT subunits have been identified in higher eukaryotic organisms (Kelleher et al., 1992; 2003; Kumar et al., 1994; Kelleher & Gilmore, 1997).     

c[D-pro-Pro-D-pro-N-methyl-Ala] adopts a rigid conformation that serves as a scaffold to mimic reverse-turns

Naturally occurring cyclic tetrapeptides (CTPs) such as tentoxin (Halloin et al., Plant Physiol 1970, 45, 310-314; Saad, Phytopathology 1970, 60, 415-418), ampicidin (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147), HC-toxin (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447), and trapoxin (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328; Itazaki et al., J Antibiot (Tokyo) 1990, 43, 1524-1532) have a wide range of biological activity and potential use ranging from herbicides (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447; Judson, J Agric Food Chem 1987, 35, 451-456) to therapeutics (Loiseau, Biopolymers 2003, 69, 363-385) for malaria (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147) and cancer (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328). To elucidate scaffolds that have few low-energy conformations and could serve as semirigid reverse-turn mimetics, the flexibility of CTPs was determined computationally. Four analogs of cyclic tetraproline c[Pro-pro-Pro-pro] with alternating L- and D-prolines, namely c[pro-Pro-pro-NMe-Ala], c[pip-Pro-pip-Pro], c[pro-Pip-pro-Pro], and c[Ala-Pro-pip-Pro] were synthesized and characterized by NOESY NMR. Both molecular mechanics and Density Functional Theory quantum calculations found these head-to-tail CTPs to be constrained to one or two relatively stable conformations. NMR structures, while not always yielding the same lowest energy conformation as expected by in silico predictions, confirmed only one or two highly populated solution conformations for all four peptides examined. c[pro-Pro-pro-NMe-Ala] was shown to have a single all trans-amide bond conformation from both in silico predictions and NMR characterization, and to be a reverse-turn mimetic by overlapping four Calpha-Cbeta bonds with those for approximately 6.5% (Tran, J Comput Aided Mol Des 2005, 19, 551-566) of reverse-turns in the Protein Data Bank PDB with a RMSD of 0.57 A.     

TASSER-based refinement of NMR structures

The TASSER structure prediction algorithm is employed to investigate whether NMR structures can be moved closer to their corresponding X-ray counterparts by automatic refinement procedures. The benchmark protein dataset includes 61 nonhomologous proteins whose structures have been determined by both NMR and X-ray experiments. Interestingly, by starting from NMR structures, the majority (79%) of TASSER refined models show a structural shift toward their X-ray structures. On average, the TASSER refined models have a root-mean-square-deviation (RMSD) from the X-ray structure of 1.785 A (1.556 A) over the entire chain (aligned region), while the average RMSD between NMR and X-ray structures (RMSD(NMR_X-ray)) is 2.080 A (1.731 A). For all proteins having a RMSD(NMR_X-ray) >2 A, the TASSER refined structures show consistent improvement. However, for the 34 proteins with a RMSD(NMR_X-ray) <2 A, there are only 21 cases (60%) where the TASSER model is closer to the X-ray structure than NMR, which may be due to the inherent resolution of TASSER. We also compare the TASSER models with 12 NMR models in the RECOORD database that have been recalculated recently by Nederveen et al. from original NMR restraints using the newest molecular dynamics tools. In 8 of 12 cases, TASSER models show a smaller RMSD to X-ray structures; in 3 of 12 cases, where RMSD(NMR_X-ray) <1 A, RECOORD does better than TASSER. These results suggest that TASSER can be a useful tool to improve the quality of NMR structures.     

Alternative structures for alternating poly(dA-dT) tracts: the structure of the B-DNA decamer C-G-A-T-A-T-A-T-C-G.

The X-ray crystal structure of the decamer C-G-A-T-A-T-A-T-C-G has been solved with two contrasting cations, Ca2+ and Mg2+. Crystals with calcium are space group P2(1)2(1)2(1), cell dimensions a = 38.76 A, b = 40.06 A, and c = 33.73 A, and diffract to 1.7-A resolution. Crystals with magnesium have the same space group, cell dimensions a = 38.69 A, b = 39.56 A, and c = 33.64 A, and diffract to 2.0 A. Their structures were solved independently by molecular replacement, beginning with idealized Arnott B-DNA geometry. The calcium structure refined to R = 17.8% for the 3683 reflections greater than 2 sigma, with 404 DNA atoms, 95 solvent peaks, and 1 Ca(H2O)7(2+) ion. The magnesium structure refined to R = 16.5% for the 1852 reflections greater than 2 sigma, with 404 DNA atoms, 62 solvent peaks, and 1 Mg(H2O)6(2+) ion. The two structures are virtually identical and are isostructural with C-G-A-T-C-G-A-T-C-G [Grzeskowiak et al. (1991) J. Biol. Chem. 266, 8861-8883] and C-G-A-T-T-A-A-T-C-G [Quintana et al. (1992) J. Mol. Biol. 225, 375-395]. Comparison of C-G-A-T-A-T-A-T-C-G with C-G-C-A-T-A-T-A-T-G-C-G [Yoon et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6332-6336] shows that the expected alternation of twist angles is found in the central A-T-A-T-A-T region of the decamer (A-T small, T-A large), but the minor groove remains wide at the center, rather than narrow. Minor groove narrowing is produced, in these two structures, not by overwinding of the helix, but by an increase in base pair propeller. This analysis confirms the concept that poly(dA-dT).poly(dA-dT) is polymorphous, with different local conformations possible in different local environments.     

HOMSTRAD: adding sequence information to structure-based alignments of homologous protein families

summary: We describe an extension to the Homologous Structure Alignment Database (HOMSTRAD; Mizuguchi et al., Protein Sci., 7, 2469-2471, 1998a) to include homologous sequences derived from the protein families database Pfam (Bateman et al., Nucleic Acids Res., 28, 263-266, 2000). HOMSTRAD is integrated with the server FUGUE (Shi et al., submitted, 2001) for recognition and alignment of homologues, benefitting from the combination of abundant sequence information and accurate structure-based alignments. AVAILABILITY The HOMSTRAD database is available at: http://www-cryst.bioc.cam.ac.uk/homstrad/. Query sequences can be submitted to the homology recognition/alignment server FUGUE at: http://www-cryst.bioc.cam.ac.uk/fugue/.     

Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.     

Comparison of the NMR and X-ray structures of the HIV-1 matrix protein: evidence for conformational changes during viral assembly.

The three-dimensional solution- and solid-state structures of the human immunodeficiency virus type-1 (HIV-1) matrix protein have been determined recently in our laboratories by NMR and X-ray crystallographic methods (Massiah et al. 1994. J Mol Biol 244:198-223; Hill et al. 1996. Proc Natl Acad Sci USA 93:3099-3104). The matrix protein exists as a monomer in solution at low millimolar protein concentrations, but forms trimers in three different crystal lattices. Although the NMR and X-ray structures are similar, detailed comparisons have revealed an approximately 6 A displacement of a short 3(10) helix (Pro 66-Gly 71) located at the trimer interface. High quality electron density and nuclear Overhauser effect (NOE) data support the integrity of the X-ray and NMR models, respectively. Because matrix apparently associates with the viral membrane as a trimer, displacement of the 3(10) helix may reflect a physiologically relevant conformational change that occurs during virion assembly and disassembly. These findings further suggest that Pro 66 and Gly 71, which bracket the 3(10) helix, serve as "hinges" that allow the 3(10) helix to undergo this structural reorientation.