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1.  MetalionRNA: computational predictor of metal-binding sites in RNA structures 
Bioinformatics  2011;28(2):198-205.
Motivation: Metal ions are essential for the folding of RNA molecules into stable tertiary structures and are often involved in the catalytic activity of ribozymes. However, the positions of metal ions in RNA 3D structures are difficult to determine experimentally. This motivated us to develop a computational predictor of metal ion sites for RNA structures.
Results: We developed a statistical potential for predicting positions of metal ions (magnesium, sodium and potassium), based on the analysis of binding sites in experimentally solved RNA structures. The MetalionRNA program is available as a web server that predicts metal ions for RNA structures submitted by the user.
Availability: The MetalionRNA web server is accessible at http://metalionrna.genesilico.pl/.
Contact: iamb@genesilico.pl
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr636
PMCID: PMC3259437  PMID: 22110243
2.  GeneSilico protein structure prediction meta-server 
Nucleic Acids Research  2003;31(13):3305-3307.
Rigorous assessments of protein structure prediction have demonstrated that fold recognition methods can identify remote similarities between proteins when standard sequence search methods fail. It has been shown that the accuracy of predictions is improved when refined multiple sequence alignments are used instead of single sequences and if different methods are combined to generate a consensus model. There are several meta-servers available that integrate protein structure predictions performed by various methods, but they do not allow for submission of user-defined multiple sequence alignments and they seldom offer confidentiality of the results. We developed a novel WWW gateway for protein structure prediction, which combines the useful features of other meta-servers available, but with much greater flexibility of the input. The user may submit an amino acid sequence or a multiple sequence alignment to a set of methods for primary, secondary and tertiary structure prediction. Fold-recognition results (target-template alignments) are converted into full-atom 3D models and the quality of these models is uniformly assessed. A consensus between different FR methods is also inferred. The results are conveniently presented on-line on a single web page over a secure, password-protected connection. The GeneSilico protein structure prediction meta-server is freely available for academic users at http://genesilico.pl/meta.
PMCID: PMC168964  PMID: 12824313
3.  COLORADO3D, a web server for the visual analysis of protein structures 
Nucleic Acids Research  2004;32(Web Server issue):W586-W589.
COLORADO3D is a World Wide Web server for the visual presentation of three-dimensional (3D) protein structures. COLORADO3D indicates the presence of potential errors (detected by ANOLEA, PROSAII, PROVE or VERIFY3D), identifies buried residues and depicts sequence conservations. As input, the server takes a file of Protein Data Bank (PDB) coordinates and, optionally, a multiple sequence alignment. As output, the server returns a PDB-formatted file, replacing the B-factor column with values of the chosen parameter (structure quality, residue burial or conservation). Thus, the coordinates of the analyzed protein ‘colored’ by COLORADO3D can be conveniently displayed with structure viewers such as RASMOL in order to visualize the 3D clusters of regions with common features, which may not necessarily be adjacent to each other at the amino acid sequence level. In particular, COLORADO3D may serve as a tool to judge a structure's quality at various stages of the modeling and refinement (during both experimental structure determination and homology modeling). The GeneSilico group used COLORADO3D in the fifth Critical Assessment of Techniques for Protein Structure Prediction (CASP5) to successfully identify well-folded parts of preliminary homology models and to guide the refinement of misthreaded protein sequences. COLORADO3D is freely available for academic use at http://asia.genesilico.pl/colorado3d/.
doi:10.1093/nar/gkh440
PMCID: PMC441578  PMID: 15215456
4.  RIBER/DIBER: a software suite for crystal content analysis in the studies of protein–nucleic acid complexes 
Bioinformatics  2012;28(6):880-881.
Summary: Co-crystallization experiments of proteins with nucleic acids do not guarantee that both components are present in the crystal. We have previously developed DIBER to predict crystal content when protein and DNA are present in the crystallization mix. Here, we present RIBER, which should be used when protein and RNA are in the crystallization drop. The combined RIBER/DIBER suite builds on machine learning techniques to make reliable, quantitative predictions of crystal content for non-expert users and high-throughput crystallography.
Availability: The program source code, Linux binaries and a web server are available at http://diber.iimcb.gov.pl/ RIBER/DIBER requires diffraction data to at least 3.0 Å resolution in MTZ or CIF (web server only) format. The RIBER/DIBER code is subject to the GNU Public License.
Contact: gchojnowski@genesilico.pl
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/bts003
PMCID: PMC3307108  PMID: 22238259
5.  Assessing the Accuracy of Template-Based Structure Prediction Metaservers by Comparison with Structural Genomics Structures 
The explosion of the size of the universe of known protein sequences has stimulated two complementary approaches to structural mapping of these sequences: theoretical structure prediction and experimental determination by structural genomics (SG). In this work, we assess the accuracy of structure prediction by two automated template-based structure prediction metaservers (genesilico.pl and bioinfo.pl) by measuring the structural similarity of the predicted models to corresponding experimental models determined a posteriori. Of 199 targets chosen from SG programs, the metaservers predicted the structures of about a fourth of them “correctly.” (In this case, “correct” was defined as placing more than 70% of the alpha carbon atoms in the model within 2 Å of the experimentally determined positions.) Almost all of the targets that could be modeled to this accuracy were those with an available template in the Protein Data Bank (PDB) with more than 25% sequence identity. The majority of those SG targets with lower sequence identity to structures in the PDB were not predicted by the metaservers with this accuracy. We also compared metaserver results to CASP8 results, finding that the models obtained by participants in the CASP competition were significantly better than those produced by the metaservers.
doi:10.1007/s10969-012-9146-2
PMCID: PMC4105012  PMID: 23086054
6.  PSPP: A Protein Structure Prediction Pipeline for Computing Clusters 
PLoS ONE  2009;4(7):e6254.
Background
Protein structures are critical for understanding the mechanisms of biological systems and, subsequently, for drug and vaccine design. Unfortunately, protein sequence data exceed structural data by a factor of more than 200 to 1. This gap can be partially filled by using computational protein structure prediction. While structure prediction Web servers are a notable option, they often restrict the number of sequence queries and/or provide a limited set of prediction methodologies. Therefore, we present a standalone protein structure prediction software package suitable for high-throughput structural genomic applications that performs all three classes of prediction methodologies: comparative modeling, fold recognition, and ab initio. This software can be deployed on a user's own high-performance computing cluster.
Methodology/Principal Findings
The pipeline consists of a Perl core that integrates more than 20 individual software packages and databases, most of which are freely available from other research laboratories. The query protein sequences are first divided into domains either by domain boundary recognition or Bayesian statistics. The structures of the individual domains are then predicted using template-based modeling or ab initio modeling. The predicted models are scored with a statistical potential and an all-atom force field. The top-scoring ab initio models are annotated by structural comparison against the Structural Classification of Proteins (SCOP) fold database. Furthermore, secondary structure, solvent accessibility, transmembrane helices, and structural disorder are predicted. The results are generated in text, tab-delimited, and hypertext markup language (HTML) formats. So far, the pipeline has been used to study viral and bacterial proteomes.
Conclusions
The standalone pipeline that we introduce here, unlike protein structure prediction Web servers, allows users to devote their own computing assets to process a potentially unlimited number of queries as well as perform resource-intensive ab initio structure prediction.
doi:10.1371/journal.pone.0006254
PMCID: PMC2707601  PMID: 19606223
7.  FunFOLD: an improved automated method for the prediction of ligand binding residues using 3D models of proteins 
BMC Bioinformatics  2011;12:160.
Background
The accurate prediction of ligand binding residues from amino acid sequences is important for the automated functional annotation of novel proteins. In the previous two CASP experiments, the most successful methods in the function prediction category were those which used structural superpositions of 3D models and related templates with bound ligands in order to identify putative contacting residues. However, whilst most of this prediction process can be automated, visual inspection and manual adjustments of parameters, such as the distance thresholds used for each target, have often been required to prevent over prediction. Here we describe a novel method FunFOLD, which uses an automatic approach for cluster identification and residue selection. The software provided can easily be integrated into existing fold recognition servers, requiring only a 3D model and list of templates as inputs. A simple web interface is also provided allowing access to non-expert users. The method has been benchmarked against the top servers and manual prediction groups tested at both CASP8 and CASP9.
Results
The FunFOLD method shows a significant improvement over the best available servers and is shown to be competitive with the top manual prediction groups that were tested at CASP8. The FunFOLD method is also competitive with both the top server and manual methods tested at CASP9. When tested using common subsets of targets, the predictions from FunFOLD are shown to achieve a significantly higher mean Matthews Correlation Coefficient (MCC) scores and Binding-site Distance Test (BDT) scores than all server methods that were tested at CASP8. Testing on the CASP9 set showed no statistically significant separation in performance between FunFOLD and the other top server groups tested.
Conclusions
The FunFOLD software is freely available as both a standalone package and a prediction server, providing competitive ligand binding site residue predictions for expert and non-expert users alike. The software provides a new fully automated approach for structure based function prediction using 3D models of proteins.
doi:10.1186/1471-2105-12-160
PMCID: PMC3123233  PMID: 21575183
8.  DARS-RNP and QUASI-RNP: New statistical potentials for protein-RNA docking 
BMC Bioinformatics  2011;12:348.
Background
Protein-RNA interactions play fundamental roles in many biological processes. Understanding the molecular mechanism of protein-RNA recognition and formation of protein-RNA complexes is a major challenge in structural biology. Unfortunately, the experimental determination of protein-RNA complexes is tedious and difficult, both by X-ray crystallography and NMR. For many interacting proteins and RNAs the individual structures are available, enabling computational prediction of complex structures by computational docking. However, methods for protein-RNA docking remain scarce, in particular in comparison to the numerous methods for protein-protein docking.
Results
We developed two medium-resolution, knowledge-based potentials for scoring protein-RNA models obtained by docking: the quasi-chemical potential (QUASI-RNP) and the Decoys As the Reference State potential (DARS-RNP). Both potentials use a coarse-grained representation for both RNA and protein molecules and are capable of dealing with RNA structures with posttranscriptionally modified residues. We compared the discriminative power of DARS-RNP and QUASI-RNP for selecting rigid-body docking poses with the potentials previously developed by the Varani and Fernandez groups.
Conclusions
In both bound and unbound docking tests, DARS-RNP showed the highest ability to identify native-like structures. Python implementations of DARS-RNP and QUASI-RNP are freely available for download at http://iimcb.genesilico.pl/RNP/
doi:10.1186/1471-2105-12-348
PMCID: PMC3179970  PMID: 21851628
RNA; protein; RNP; macromolecular docking; complex modeling; structural bioinformatics
9.  Automated 3D structure composition for large RNAs 
Nucleic Acids Research  2012;40(14):e112.
Understanding the numerous functions that RNAs play in living cells depends critically on knowledge of their three-dimensional structure. Due to the difficulties in experimentally assessing structures of large RNAs, there is currently great demand for new high-resolution structure prediction methods. We present the novel method for the fully automated prediction of RNA 3D structures from a user-defined secondary structure. The concept is founded on the machine translation system. The translation engine operates on the RNA FRABASE database tailored to the dictionary relating the RNA secondary structure and tertiary structure elements. The translation algorithm is very fast. Initial 3D structure is composed in a range of seconds on a single processor. The method assures the prediction of large RNA 3D structures of high quality. Our approach needs neither structural templates nor RNA sequence alignment, required for comparative methods. This enables the building of unresolved yet native and artificial RNA structures. The method is implemented in a publicly available, user-friendly server RNAComposer. It works in an interactive mode and a batch mode. The batch mode is designed for large-scale modelling and accepts atomic distance restraints. Presently, the server is set to build RNA structures of up to 500 residues.
doi:10.1093/nar/gks339
PMCID: PMC3413140  PMID: 22539264
10.  CING: an integrated residue-based structure validation program suite 
Journal of Biomolecular Nmr  2012;54(3):267-283.
We present a suite of programs, named CING for Common Interface for NMR Structure Generation that provides for a residue-based, integrated validation of the structural NMR ensemble in conjunction with the experimental restraints and other input data. External validation programs and new internal validation routines compare the NMR-derived models with empirical data, measured chemical shifts, distance- and dihedral restraints and the results are visualized in a dynamic Web 2.0 report. A red–orange–green score is used for residues and restraints to direct the user to those critiques that warrant further investigation. Overall green scores below ~20 % accompanied by red scores over ~50 % are strongly indicative of poorly modelled structures. The publically accessible, secure iCing webserver (https://nmr.le.ac.uk) allows individual users to upload the NMR data and run a CING validation analysis.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-012-9669-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-012-9669-7
PMCID: PMC3483101  PMID: 22986687
NMR; Structure validation; PDB; Errors; Quality; Protein structure
11.  RNA Structure Determination Using SAXS Data 
The journal of physical chemistry. B  2010;114(31):10039-10048.
Exploiting the experimental information from small-angle x-ray solution scattering (SAXS) in conjunction with structure prediction algorithms can be advantageous in the case of ribonucleic acids (RNA), where global restraints on the 3D fold are often lacking. Traditional usage of SAXS data often starts by attempting to reconstruct the molecular shape ab initio, which is subsequently used to assess the quality of model Here, an alternative strategy is explored whereby the models from a very large decoy set are directly sorted according to their fit to the SAXS data is developed. For rapid computation of SAXS patterns, the method developed here makes use of a coarse-grained representation of RNA. It also accounts for the explicit treatment of the contribution to the scattering of water molecules and ions surrounding the RNA. The method, called Fast-SAXS-RNA, is first calibrated using a transfer RNA (tRNA-val) and then tested on the P4-P6 fragment of group I intron (P4-P6). Fast-SAXS-RNA is then used as a filter for decoy models generated by the MC-Fold and MC-Sym pipeline, a suite of RNA 3D all-atoms structure algorithms that encode and exploit RNA 3D architectural principles. The ability of Fast-SAXS-RNA to discriminate native folds is tested against three widely used RNA molecules in molecular modeling benchmarks: the tRNA, the P4-P6, and a synthetic hairpin suspected to assemble into a homodimer. For each molecule, a large pool of decoys are generated, scored, and ranked using Fast-SAXS-RNA. The method is able to identify low-RMSD models among top ranking structures, for both tRNA and P4-P6. For the hairpin, the approach correctly identifies the dimeric state as the solution structure over the monomeric state and alternative secondary structures. The method offers a powerful strategy for recognizing native RNA conformations as well as multimeric assemblies and alternative secondary structures, thus enabling high-throughput RNA structure determination using SAXS data.
doi:10.1021/jp1057308
PMCID: PMC3164809  PMID: 20684627
12.  Protein-Protein Docking with F2Dock 2.0 and GB-Rerank 
PLoS ONE  2013;8(3):e51307.
Motivation
Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA) based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error.
Results
The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other.
Availability
The docking protocol has been implemented as a server with a graphical client (TexMol) which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http://www.cs.utexas.edu/~bajaj/cvc/software/f2dock.shtml. Client: http://www.cs.utexas.edu/~bajaj/cvc/software/f2dockclient.shtml.
doi:10.1371/journal.pone.0051307
PMCID: PMC3590208  PMID: 23483883
13.  MODOMICS: a database of RNA modification pathways. 2008 update 
Nucleic Acids Research  2008;37(Database issue):D118-D121.
MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.
doi:10.1093/nar/gkn710
PMCID: PMC2686465  PMID: 18854352
14.  The utility of geometrical and chemical restraint information extracted from predicted ligand binding sites in protein structure refinement 
Journal of structural biology  2010;173(3):558-569.
Exhaustive exploration of molecular interactions at the level of complete proteomes requires efficient and reliable computational approaches to protein function inference. Ligand docking and ranking techniques show considerable promise in their ability to quantify the interactions between proteins and small molecules. Despite the advances in the development of docking approaches and scoring functions, the genome-wide application of many ligand docking/screening algorithms is limited by the quality of the binding sites in theoretical receptor models constructed by protein structure prediction. In this study, we describe a new template-based method for the local refinement of ligand-binding regions in protein models using remotely related templates identified by threading. We designed a Support Vector Regression (SVR) model that selects correct binding site geometries in a large ensemble of multiple receptor conformations. The SVR model employs several scoring functions that impose geometrical restraints on the Cα positions, account for the specific chemical environment within a binding site and optimize the interactions with putative ligands. The SVR score is well correlated with the RMSD from the native structure; in 47% (70%) of the cases, the Pearson’s correlation coefficient is >0.5 (>0.3). When applied to weakly homologous models, the average heavy atom, local RMSD from the native structure of the top-ranked (best of top five) binding site geometries is 3.1 Å (2.9 Å) for roughly half of the targets; this represents a 0.1 (0.3) Å average improvement over the original predicted structure. Focusing on the subset of strongly conserved residues, the average heavy atom RMSD is 2.6 Å (2.3 Å). Furthermore, we estimate the upper bound of template-based binding site refinement using only weakly related proteins to be ~2.6 Å RMSD. This value also corresponds to the plasticity of the ligand-binding regions in distant homologues. The Binding Site Refinement (BSR) approach is available to the scientific community as a web server that can be accessed at http://cssb.biology.gatech.edu/bsr/.
doi:10.1016/j.jsb.2010.09.009
PMCID: PMC3036769  PMID: 20850544
Ligand-binding site refinement; proteinthreading; protein structure prediction; ligand-binding site prediction; ensemble docking; molecular function
15.  RNA Bricks—a database of RNA 3D motifs and their interactions 
Nucleic Acids Research  2013;42(Database issue):D123-D131.
The RNA Bricks database (http://iimcb.genesilico.pl/rnabricks), stores information about recurrent RNA 3D motifs and their interactions, found in experimentally determined RNA structures and in RNA–protein complexes. In contrast to other similar tools (RNA 3D Motif Atlas, RNA Frabase, Rloom) RNA motifs, i.e. ‘RNA bricks’ are presented in the molecular environment, in which they were determined, including RNA, protein, metal ions, water molecules and ligands. All nucleotide residues in RNA bricks are annotated with structural quality scores that describe real-space correlation coefficients with the electron density data (if available), backbone geometry and possible steric conflicts, which can be used to identify poorly modeled residues. The database is also equipped with an algorithm for 3D motif search and comparison. The algorithm compares spatial positions of backbone atoms of the user-provided query structure and of stored RNA motifs, without relying on sequence or secondary structure information. This enables the identification of local structural similarities among evolutionarily related and unrelated RNA molecules. Besides, the search utility enables searching ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions.
doi:10.1093/nar/gkt1084
PMCID: PMC3965019  PMID: 24220091
16.  AIDA: ab initio domain assembly server 
Nucleic Acids Research  2014;42(Web Server issue):W308-W313.
AIDA: ab initio domain assembly server, available at http://ffas.burnham.org/AIDA/ is a tool that can identify domains in multi-domain proteins and then predict their 3D structures and relative spatial arrangements. The server is free and open to all users, and there is an option for a user to provide an e-mail to get the link to result page. Domains are evolutionary conserved and often functionally independent units in proteins. Most proteins, especially eukaryotic ones, consist of multiple domains while at the same time, most experimentally determined protein structures contain only one or two domains. As a result, often structures of individual domains in multi-domain proteins can be accurately predicted, but the mutual arrangement of different domains remains unknown. To address this issue we have developed AIDA program, which combines steps of identifying individual domains, predicting (separately) their structures and assembling them into multiple domain complexes using an ab initio folding potential to describe domain–domain interactions. AIDA server not only supports the assembly of a large number of continuous domains, but also allows the assembly of domains inserted into other domains. Users can also provide distance restraints to guide the AIDA energy minimization.
doi:10.1093/nar/gku369
PMCID: PMC4086082  PMID: 24831546
17.  SELECTpro: effective protein model selection using a structure-based energy function resistant to BLUNDERs 
Background
Protein tertiary structure prediction is a fundamental problem in computational biology and identifying the most native-like model from a set of predicted models is a key sub-problem. Consensus methods work well when the redundant models in the set are the most native-like, but fail when the most native-like model is unique. In contrast, structure-based methods score models independently and can be applied to model sets of any size and redundancy level. Additionally, structure-based methods have a variety of important applications including analogous fold recognition, refinement of sequence-structure alignments, and de novo prediction. The purpose of this work was to develop a structure-based model selection method based on predicted structural features that could be applied successfully to any set of models.
Results
Here we introduce SELECTpro, a novel structure-based model selection method derived from an energy function comprising physical, statistical, and predicted structural terms. Novel and unique energy terms include predicted secondary structure, predicted solvent accessibility, predicted contact map, β-strand pairing, and side-chain hydrogen bonding.
SELECTpro participated in the new model quality assessment (QA) category in CASP7, submitting predictions for all 95 targets and achieved top results. The average difference in GDT-TS between models ranked first by SELECTpro and the most native-like model was 5.07. This GDT-TS difference was less than 1% of the GDT-TS of the most native-like model for 18 targets, and less than 10% for 66 targets. SELECTpro also ranked the single most native-like first for 15 targets, in the top five for 39 targets, and in the top ten for 53 targets, more often than any other method. Because the ranking metric is skewed by model redundancy and ignores poor models with a better ranking than the most native-like model, the BLUNDER metric is introduced to overcome these limitations. SELECTpro is also evaluated on a recent benchmark set of 16 small proteins with large decoy sets of 12500 to 20000 models for each protein, where it outperforms the benchmarked method (I-TASSER).
Conclusion
SELECTpro is an effective model selection method that scores models independently and is appropriate for use on any model set. SELECTpro is available for download as a stand alone application at: . SELECTpro is also available as a public server at the same site.
doi:10.1186/1472-6807-8-52
PMCID: PMC2667183  PMID: 19055744
18.  Protein–protein HADDocking using exclusively pseudocontact shifts 
Journal of Biomolecular Nmr  2011;50(3):263-266.
In order to enhance the structure determination process of macromolecular assemblies by NMR, we have implemented long-range pseudocontact shift (PCS) restraints into the data-driven protein docking package HADDOCK. We demonstrate the efficiency of the method on a synthetic, yet realistic case based on the lanthanide-labeled N-terminal ε domain of the E. coli DNA polymerase III (ε186) in complex with the HOT domain. Docking from the bound form of the two partners is swiftly executed (interface RMSDs < 1 Å) even with addition of very large amount of noise, while the conformational changes of the free form still present some challenges (interface RMSDs in a 3.1–3.9 Å range for the ten lowest energy complexes). Finally, using exclusively PCS as experimental information, we determine the structure of ε186 in complex with the HOT-homologue θ subunit of the E. coli DNA polymerase III.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-011-9514-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-011-9514-4
PMCID: PMC3133697  PMID: 21626213
HADDOCK; Pseudocontact shift; Protein docking; Paramagnetic NMR; DNA polymerase III
19.  Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution 
Recent developments in PHENIX are reported that allow the use of reference-model torsion restraints, secondary-structure hydrogen-bond restraints and Ramachandran restraints for improved macromolecular refinement in phenix.refine at low resolution.
Traditional methods for macromolecular refinement often have limited success at low resolution (3.0–3.5 Å or worse), producing models that score poorly on crystallographic and geometric validation criteria. To improve low-resolution refinement, knowledge from macromolecular chemistry and homology was used to add three new coordinate-restraint functions to the refinement program phenix.refine. Firstly, a ‘reference-model’ method uses an identical or homologous higher resolution model to add restraints on torsion angles to the geometric target function. Secondly, automatic restraints for common secondary-structure elements in proteins and nucleic acids were implemented that can help to preserve the secondary-structure geometry, which is often distorted at low resolution. Lastly, we have implemented Ramachandran-based restraints on the backbone torsion angles. In this method, a ϕ,ψ term is added to the geometric target function to minimize a modified Ramachandran landscape that smoothly combines favorable peaks identified from non­redundant high-quality data with unfavorable peaks calculated using a clash-based pseudo-energy function. All three methods show improved MolProbity validation statistics, typically complemented by a lowered R free and a decreased gap between R work and R free.
doi:10.1107/S0907444911047834
PMCID: PMC3322597  PMID: 22505258
macromolecular crystallography; low resolution; refinement; automation
20.  High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment 
Journal of Biomolecular Nmr  2007;40(1):1-13.
Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 Å from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-007-9204-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-007-9204-4
PMCID: PMC2758389  PMID: 18026911
NMR solution structure; Homodimer CylR2; Paramagnetic relaxation enhancement; PALES; Residual dipolar couplings; Rigid-body docking
21.  Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure 
Journal of Biomolecular Nmr  2012;53(4):321-339.
Restrained molecular dynamics simulations are a robust, though perhaps underused, tool for the end-stage refinement of biomolecular structures. We demonstrate their utility—using modern simulation protocols, optimized force fields, and inclusion of explicit solvent and mobile counterions—by re-investigating the solution structures of two RNA hairpins that had previously been refined using conventional techniques. The structures, both domain 5 group II intron ribozymes from yeast ai5γ and Pylaiella littoralis, share a nearly identical primary sequence yet the published 3D structures appear quite different. Relatively long restrained MD simulations using the original NMR restraint data identified the presence of a small set of violated distance restraints in one structure and a possibly incorrect trapped bulge nucleotide conformation in the other structure. The removal of problematic distance restraints and the addition of a heating step yielded representative ensembles with very similar 3D structures and much lower pairwise RMSD values. Analysis of ion density during the restrained simulations helped to explain chemical shift perturbation data published previously. These results suggest that restrained MD simulations, with proper caution, can be used to “update” older structures or aid in the refinement of new structures that lack sufficient experimental data to produce a high quality result. Notable cautions include the need for sufficient sampling, awareness of potential force field bias (such as small angle deviations with the current AMBER force fields), and a proper balance between the various restraint weights.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-012-9642-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-012-9642-5
PMCID: PMC3405240  PMID: 22714631
RNA structure; Molecular dynamics; Residual dipolar coupling restraints; Bulge structure; Force fields; Ion binding
22.  CPORT: A Consensus Interface Predictor and Its Performance in Prediction-Driven Docking with HADDOCK 
PLoS ONE  2011;6(3):e17695.
Background
Macromolecular complexes are the molecular machines of the cell. Knowledge at the atomic level is essential to understand and influence their function. However, their number is huge and a significant fraction is extremely difficult to study using classical structural methods such as NMR and X-ray crystallography. Therefore, the importance of large-scale computational approaches in structural biology is evident. This study combines two of these computational approaches, interface prediction and docking, to obtain atomic-level structures of protein-protein complexes, starting from their unbound components.
Methodology/Principal Findings
Here we combine six interface prediction web servers into a consensus method called CPORT (Consensus Prediction Of interface Residues in Transient complexes). We show that CPORT gives more stable and reliable predictions than each of the individual predictors on its own. A protocol was developed to integrate CPORT predictions into our data-driven docking program HADDOCK. For cases where experimental information is limited, this prediction-driven docking protocol presents an alternative to ab initio docking, the docking of complexes without the use of any information. Prediction-driven docking was performed on a large and diverse set of protein-protein complexes in a blind manner. Our results indicate that the performance of the HADDOCK-CPORT combination is competitive with ZDOCK-ZRANK, a state-of-the-art ab initio docking/scoring combination. Finally, the original interface predictions could be further improved by interface post-prediction (contact analysis of the docking solutions).
Conclusions/Significance
The current study shows that blind, prediction-driven docking using CPORT and HADDOCK is competitive with ab initio docking methods. This is encouraging since prediction-driven docking represents the absolute bottom line for data-driven docking: any additional biological knowledge will greatly improve the results obtained by prediction-driven docking alone. Finally, the fact that original interface predictions could be further improved by interface post-prediction suggests that prediction-driven docking has not yet been pushed to the limit. A web server for CPORT is freely available at http://haddock.chem.uu.nl/services/CPORT.
doi:10.1371/journal.pone.0017695
PMCID: PMC3064578  PMID: 21464987
23.  JLigand: a graphical tool for the CCP4 template-restraint library 
The CCP4 template-restraint library defines restraints for biopolymers, their modifications and ligands that are used in macromolecular structure refinement. JLigand is a graphical editor for generating descriptions of new ligands and covalent linkages.
Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user’s additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user’s library and generate new monomer entries is described, as well as new entries for links and associated modifications.
doi:10.1107/S090744491200251X
PMCID: PMC3322602  PMID: 22505263
macromolecular refinement; restraint library; molecular graphics
24.  NRG-CING: integrated validation reports of remediated experimental biomolecular NMR data and coordinates in wwPDB 
Nucleic Acids Research  2011;40(Database issue):D519-D524.
For many macromolecular NMR ensembles from the Protein Data Bank (PDB) the experiment-based restraint lists are available, while other experimental data, mainly chemical shift values, are often available from the BioMagResBank. The accuracy and precision of the coordinates in these macromolecular NMR ensembles can be improved by recalculation using the available experimental data and present-day software. Such efforts, however, generally fail on half of all NMR ensembles due to the syntactic and semantic heterogeneity of the underlying data and the wide variety of formats used for their deposition. We have combined the remediated restraint information from our NMR Restraints Grid (NRG) database with available chemical shifts from the BioMagResBank and the Common Interface for NMR structure Generation (CING) structure validation reports into the weekly updated NRG-CING database (http://nmr.cmbi.ru.nl/NRG-CING). Eleven programs have been included in the NRG-CING production pipeline to arrive at validation reports that list for each entry the potential inconsistencies between the coordinates and the available experimental NMR data. The longitudinal validation of these data in a publicly available relational database yields a set of indicators that can be used to judge the quality of every macromolecular structure solved with NMR. The remediated NMR experimental data sets and validation reports are freely available online.
doi:10.1093/nar/gkr1134
PMCID: PMC3245154  PMID: 22139937
25.  An NMR-based scoring function improves the accuracy of binding pose predictions by docking by two orders of magnitude 
Journal of Biomolecular Nmr  2011;52(1):23-30.
Low-affinity ligands can be efficiently optimized into high-affinity drug leads by structure based drug design when atomic-resolution structural information on the protein/ligand complexes is available. In this work we show that the use of a few, easily obtainable, experimental restraints improves the accuracy of the docking experiments by two orders of magnitude. The experimental data are measured in nuclear magnetic resonance spectra and consist of protein-mediated NOEs between two competitively binding ligands. The methodology can be widely applied as the data are readily obtained for low-affinity ligands in the presence of non-labelled receptor at low concentration. The experimental inter-ligand NOEs are efficiently used to filter and rank complex model structures that have been pre-selected by docking protocols. This approach dramatically reduces the degeneracy and inaccuracy of the chosen model in docking experiments, is robust with respect to inaccuracy of the structural model used to represent the free receptor and is suitable for high-throughput docking campaigns.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-011-9590-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-011-9590-5
PMCID: PMC3266494  PMID: 22167466
NMR; INPHARMA; NOE; Docking; Drug design

Results 1-25 (1042672)