Satisfaction Of Spatial Restraints Research Articles

Revisiting the "satisfaction of spatial restraints" approach of MODELLER for protein homology modeling.

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the "modeling by satisfaction of spatial restraints" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of σ values profoundly influences 3D modeling. We show that the σ values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the program's predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLER's objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance.

Studies on computational grafting of malarial epitopes in serum albumin

BackgroundAntigens (or their epitopes)-carrier protein combinations are extensively utilized for vaccine development strategies. Chemical conjugation methods are cumbersome with limited conjugation sites. Computational protein modelling methods can evaluate every position of a carrier protein for conjugation via epitope grafting in a reasonable time frame as compared to wet experimental techniques. Graftibility of positions can be estimated by the presence of native atomic contacts in resulting chimeric antigen. MethodologyFive epitopes were selected, and computational grafting at each position was performed in three templates of serum albumin. Protein modelling algorithms such as segment matching and satisfaction of spatial restraints were employed for computational grafting. Contact-based protein discriminatory function was used to evaluate the chimeric proteins having native atomic contacts. ResultsOn the evaluation of approximately 1 million distinct protein modelling simulations, region around the 450th position of serum albumin was observed to be suitable for epitope grafting. ConclusionComputational protein modelling tools may be used to design a chimeric antigen. The approach may overcome the limitations associated with chemical conjugation and furthermore harness the potential of custom gene synthesis/recombinant protein production.

Computational Toolset for Glycoconjugate Modeling and Simulation

Characterizing glycoconjugates and protein-glycan interactions in the context of three-dimensional structures is important in understanding glycan's biological roles and develop efficient therapeutic agents. However, it is challenging to achieve this goal due to limitations in available experimental approaches and computational tools. To tackle this problem, we have developed GlycanStructure.ORG, a web portal to provide various computational tools. (1) Glycan Reader has been developed for automatic detection and annotation of carbohydrates, their chemical derivatives, and glycosidic linkages from PDB files (both in the PDB and mmCIF formats). Molecular system and input generations for the glycoconjugate simulation are available through CHARMM-GUI, http://www.charmm-gui.org. (2) The glycan fragment database (GFDB) provides an intuitive glycan sequence search tool that allows users to search for complex glycan structures in the PDB. After a glycan search, each selected glycosidic torsion angle distribution can be displayed and clustering analysis is used to provide most populated glycan structures, which can be useful for glycan structure modeling. (3) GS-align is an algorithm to identify the best structural alignment between glycan structure pairs and to provide a length-independent score of the structural similarity. This tool is useful to study the relationship between multiple glycoforms and biological functions, and glycan structure prediction. (4) Glycan Modeler is a template-based method to predict glycan structures from a given sequence using the PDB glycan structures as fragment template. After templates are identified by the random forest algorithm, Glycan Modeler provides two conformational sampling approaches that are assembly of rigid bodies and satisfaction of spatial restraints using by conformational space annealing (CSA).

Multi-State Modeling of Biomolecules by Satisfaction of Spatial Restraints from Single-Particle Electron Microscopy Images

Single proteins and macromolecular complexes can exist in multiple conformational and compositional states. Accurate modeling of these ensembles of states is key to understanding and modulating the molecular function. Single-particle electron microscopy (EM) can produce a wealth of structural information. Here, we describe an integrative method for computing a multi-state model of a biomolecular system, based primarily on input particle images from EM. The approach also leverages prior knowledge about the stereochemistry of molecules, image noise, and potentially other sources of information.

Assembly of macromolecular complexes by satisfaction of spatial restraints from electron microscopy images

To obtain a structural model of a macromolecular assembly by single-particle EM, a large number of particle images need to be collected, aligned, clustered, averaged, and finally assembled via reconstruction into a 3D density map. This process is limited by the number and quality of the particle images, the accuracy of the initial model, and the compositional and conformational heterogeneity. Here, we describe a structure determination method that avoids the reconstruction procedure. The atomic structures of the individual complex components are assembled by optimizing a match against 2D EM class-average images, an excluded volume criterion, geometric complementarity, and optional restraints from proteomics and chemical cross-linking experiments. The optimization relies on a simulated annealing Monte Carlo search and a divide-and-conquer message-passing algorithm. Using simulated and experimentally determined EM class averages for 12 and 4 protein assemblies, respectively, we show that a few class averages can indeed result in accurate models for complexes of as many as five subunits. Thus, integrative structural biology can now benefit from the relative ease with which the EM class averages are determined.

Homology modeling studies of beta(1,3)‐D‐glucan synthase of Moniliophthora perniciosa

AbstractThe Witches' Broom Disease, caused by the hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, drastically reduced the production of cocoa in Brazil. Phytosanitation, chemical control, genetically resistant strains, and biological control still leave flaws in the disease eradication process. Effort has been expended in the elucidation of molecular targets, in particular, the structural components of the fungal cell wall, such as the beta(1,3)‐D‐glucan synthase. This enzyme is essential for the cellular construction of the wall, as it catalyses the formation of beta(1,3)‐D‐glucans. Protein structure homology modeling approaches are able to determine the structure of proteins without performing experimental steps, considering the barriers related to experimental methods for the structural determination of molecular targets. The presence of the conserved catalytic residues in members of the same glycosyltransferase family and overall structural analysis suggests that they catalyze glycosyl transfer reactions by similar mechanisms. Therefore, the objective of this study was to determine the three‐dimensional model of the enzyme beta(1,3)‐D‐glucan synthase of M. perniciosa by homology modeling. Both procedures were performed to build the models: a comparative modeling by satisfaction of spatial restraints in MODELLER and a modeling by assembly of rigid bodies in the SWISS‐MODEL software. The models were elected based on analysis of the stereochemistry quality and a quantitative assessment of similarity from the obtained models and to templates. A reasonable structural model was obtained of the beta(1,3)‐D‐glucan synthase enzyme (BegS1). The BegS1 model showed two distinct α/β domains, features of the inverting glycosyltransferase family, and the topology of the folded structure showed 7 beta‐strands and 13 alpha‐helices. The BegS1 model showed the presence of a catalytic cavity formed by the conserved aspartic acid residues (Asp326, 345, 353, and 354 DDxD motif) implicated in substrate binding and/or catalysis. In the BegS1 model, this cavity is near a loop region, as was observed in the GT‐2 family structure. It is encouraging to find that the model for BegS1 agrees well with structures from the GT‐2 enzyme family. © 2012 Wiley Periodicals, Inc.

Homology-based Identification of Capsid Determinants That Protect HIV1 from Human TRIM5α Restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.

English

The bacteria Clostridium botulinum causing botulism. Botulism also known as botulinus intoxication is a rare but serious paralytic illness caused by botulinum toxin, which is produced by the bacterium Clostridium botulinum.CDP-diacylglycerol-serine O-phosphatidyltransferase is an enzyme that catalyzes the CDP-diacylglycerol, L-serine and produce Cytidine monophosphate (CMP), (3-snphosphatidyl)-L-serine in Clostridium botulinum. CMP is a nucleotide that is helpful in RNA synthesis in Clostridium botulinum.The objective of enzyme inhibition needs structural characterization from its 3-D structure in rational structure based drug design. Homology modeling can produce high-quality structural models when the target and templates are closely related. MODELLER is the program used for homology modeling which provides accurate and efficient models to build loops and side chains found non-identical in sequence. It implements comparative protein structure modeling by satisfaction of spatial restraints. The stereo chemical quality of a best model is validated by PROCHECK server with 86.2% residues under favored region from Ramachandran plot. The CASTp is used for locating, delineating and measuring concave surface regions on three-dimensional structures of proteins. The residues are identified in burial cavity of the best model as Leu (131,141,156), Ala (132), Ile (138,142), Asn (145), Thr (149,153), and Gly (151) with hydrophobic nature. These include pocket located on protein surfaces and voids buried in the interior of proteins that are frequently associated with binding events. Thus present studies of modeling of CDP-diacylglycerol-serine O-phosphatidyltransferas enzyme has brought future prospective to fight against botulism disease and provide better health standaaards for community.

Structure determination of genomic domains by satisfaction of spatial restraints

The three-dimensional (3D) architecture of a genome is non-random and known to facilitate the spatial colocalization of regulatory elements with the genes they regulate. Determining the 3D structure of a genome may therefore probe an essential step in characterizing how genes are regulated. Currently, there are several experimental and theoretical approaches that aim at determining the 3D structure of genomes and genomic domains; however, approaches integrating experiments and computation to identify the most likely 3D folding of a genome at medium to high resolutions have not been widely explored. Here, we review existing methodologies and propose that the integrative modeling platform (http://www.integrativemodeling.org), a computational package developed for structurally characterizing protein assemblies, could be used for integrating diverse experimental data towards the determination of the 3D architecture of genomic domains and entire genomes at unprecedented resolution. Our approach, through the visualization of looping interactions between distal regulatory elements, will allow for the characterization of global chromatin features and their relation to gene expression. We illustrate our work by outlining the recent determination of the 3D architecture of the α-globin domain in the human genome.

RNA channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.

A modeled structure for amidase-03 from Bacillus anthracis.

Homology models of amidase-03 from Bacillus anthracis were constructed using Modeller (9v2). Modeller constructs protein models using an automated approach for comparative protein structure modeling by the satisfaction of spatial restraints. A template structure of Listeria monocytogenes bacteriophage PSA endolysin PlyPSA (PDB ID: 1XOV) was selected from protein databank (PDB) using BLASTp with BLOSUM62 sequence alignment scoring matrix. We generated five models using the Modeller default routine in which initial coordinates are randomized and evaluated by pseudo-energy parameters. The protein models were validated using PROCHECK and energy minimized using the steepest descent method in GROMACS 3.2 (flexible SPC water model in cubic box of size 1 Å instead of rigid SPC model). We used G43a1 force field in GROMACS for energy calculations and the generated structure was subsequently analyzed using the VMD software for stereo-chemistry, atomic clash and misfolding. A detailed analysis of the amidase-03 model structure from Bacillus anthracis will provide insight to the molecular design of suitable inhibitors as drug candidates.

AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.

Integration of Small-Angle X-Ray Scattering Data into Structural Modeling of Proteins and Their Assemblies

A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluster of many independently calculated solutions. To quantify how well the quaternary structures are determined based on their SAXS profiles, we used a benchmark of 12 simulated examples as well as an experimental SAXS profile of the homotetramer d-xylose isomerase. Optimization of the SAXS-dependent scoring function generally results in accurate models if sufficiently precise approximations for the constituent rigid bodies are available; otherwise, the best scoring models can have significant errors. Thus, SAXS profiles can play a useful role in the structural characterization of proteins and assemblies if they are combined with additional data and used judiciously. Our integration of a SAXS profile into modeling by satisfaction of spatial restraints will facilitate further integration of different kinds of data for structure determination of proteins and their assemblies.

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, <http://www-nmr.cabm.rutgers.edu/HOMA>, 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.

Refinement of Protein Structures by Iterative Comparative Modeling and CryoEM Density Fitting

We developed a method for structure characterization of assembly components by iterative comparative protein structure modeling and fitting into cryo-electron microscopy (cryoEM) density maps. Specifically, we calculate a comparative model of a given component by considering many alternative alignments between the target sequence and a related template structure while optimizing the fit of a model into the corresponding density map. The method relies on the previously developed Moulder protocol that iterates over alignment, model building, and model assessment. The protocol was benchmarked using 20 varied target-template pairs of known structures with less than 30% sequence identity and corresponding simulated density maps at resolutions from 5A to 25A. Relative to the models based on the best existing sequence profile alignment methods, the percentage of C(alpha) atoms that are within 5A of the corresponding C(alpha) atoms in the superposed native structure increases on average from 52% to 66%, which is half-way between the starting models and the models from the best possible alignments (82%). The test also reveals that despite the improvements in the accuracy of the fitness function, this function is still the bottleneck in reducing the remaining errors. To demonstrate the usefulness of the protocol, we applied it to the upper domain of the P8 capsid protein of rice dwarf virus that has been studied by cryoEM at 6.8A. The C(alpha) root-mean-square deviation of the model based on the remotely related template, bluetongue virus VP7, improved from 8.7A to 6.0A, while the best possible model has a C(alpha) RMSD value of 5.3A. Moreover, the resulting model fits better into the cryoEM density map than the initial template structure. The method is being implemented in our program MODELLER for protein structure modeling by satisfaction of spatial restraints and will be applicable to the rapidly increasing number of cryoEM density maps of macromolecular assemblies.

Structural Characterization of Assemblies from Overall Shape and Subcomplex Compositions

We suggest structure characterization of macromolecular assemblies by combining assembly shape determined by electron cyromicroscopy with information about subunit proximity determined by affinity purification. To achieve this aim, structure characterization is expressed as a problem in satisfaction of spatial restraints that (1) represents subunits as spheres, (2) encodes information about the subunit excluded volume, assembly shape, and pulldowns in a scoring function, and (3) finds subunit configurations that satisfy the input restraints by an optimization of the scoring function. Testing of the approach with model systems suggests its feasibility.

Macromolecular Assemblies Highlighted

The functional units in cells are often assemblies of macromolecules, including proteins and nucleic acids. Therefore, the knowledge of structure, dynamics, and energetics of macromolecular complexes is necessary for a mechanistic description of biochemical and cellular processes. This special issue of Structure , "Macromolecular Assemblies Highlighted," presents ten research articles and five reviews on the determination, representation, visualization, archival, and dissemination of assembly structure and dynamics.

ModLoop: automated modeling of loops in protein structures

ModLoop is a web server for automated modeling of loops in protein structures. The input is the atomic coordinates of the protein structure in the Protein Data Bank format, and the specification of the starting and ending residues of one or more segments to be modeled, containing no more than 20 residues in total. The output is the coordinates of the non-hydrogen atoms in the modeled segments. A user provides the input to the server via a simple web interface, and receives the output by e-mail. The server relies on the loop modeling routine in MODELLER that predicts the loop conformations by satisfaction of spatial restraints, without relying on a database of known protein structures. For a rapid response, ModLoop runs on a cluster of Linux PC computers. The server is freely accessible to academic users at http://salilab.org/modloop

Evolution and Physics in Comparative Protein Structure Modeling

From a physical perspective, the native structure of a protein is a consequence of physical forces acting on the protein and solvent atoms during the folding process. From a biological perspective, the native structure of proteins is a result of evolution over millions of years. Correspondingly, there are two types of protein structure prediction methods, de novo prediction and comparative modeling. We review comparative protein structure modeling and discuss the incorporation of physical considerations into the modeling process. A good starting point for achieving this aim is provided by comparative modeling by satisfaction of spatial restraints. Incorporation of physical considerations is illustrated by an inclusion of solvation effects into the modeling of loops.

Comparative protein modeling by satisfaction of spatial restraints : Andrej Ŝali, Rockefeller University, New York, NY 10021, USA

Comparative protein modeling by satisfaction of spatial restraints : Andrej Ŝali, Rockefeller University, New York, NY 10021, USA