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1.  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
2.  IsoCleft Finder – a web-based tool for the detection and analysis of protein binding-site geometric and chemical similarities 
F1000Research  2013;2:117.
IsoCleft Finder is a web-based tool for the detection of local geometric and chemical similarities between potential small-molecule binding cavities and a non-redundant dataset of ligand-bound known small-molecule binding-sites. The non-redundant dataset developed as part of this study is composed of 7339 entries representing unique Pfam/PDB-ligand (hetero group code) combinations with known levels of cognate ligand similarity. The query cavity can be uploaded by the user or detected automatically by the system using existing PDB entries as well as user-provided structures in PDB format. In all cases, the user can refine the definition of the cavity interactively via a browser-based Jmol 3D molecular visualization interface. Furthermore, users can restrict the search to a subset of the dataset using a cognate-similarity threshold. Local structural similarities are detected using the IsoCleft software and ranked according to two criteria (number of atoms in common and Tanimoto score of local structural similarity) and the associated Z-score and p-value measures of statistical significance. The results, including predicted ligands, target proteins, similarity scores, number of atoms in common, etc., are shown in a powerful interactive graphical interface. This interface permits the visualization of target ligands superimposed on the query cavity and additionally provides a table of pairwise ligand topological similarities. Similarities between top scoring ligands serve as an additional tool to judge the quality of the results obtained. We present several examples where IsoCleft Finder provides useful functional information. IsoCleft Finder results are complementary to existing approaches for the prediction of protein function from structure, rational drug design and x-ray crystallography. IsoCleft Finder can be found at: http://bcb.med.usherbrooke.ca/isocleftfinder.
doi:10.12688/f1000research.2-117.v1
PMCID: PMC3892921  PMID: 24555058
3.  IsoCleft Finder – a web-based tool for the detection and analysis of protein binding-site geometric and chemical similarities 
F1000Research  2013;2:117.
IsoCleft Finder is a web-based tool for the detection of local geometric and chemical similarities between potential small-molecule binding cavities and a non-redundant dataset of ligand-bound known small-molecule binding-sites. The non-redundant dataset developed as part of this study is composed of 7339 entries representing unique Pfam/PDB-ligand (hetero group code) combinations with known levels of cognate ligand similarity. The query cavity can be uploaded by the user or detected automatically by the system using existing PDB entries as well as user-provided structures in PDB format. In all cases, the user can refine the definition of the cavity interactively via a browser-based Jmol 3D molecular visualization interface. Furthermore, users can restrict the search to a subset of the dataset using a cognate-similarity threshold. Local structural similarities are detected using the IsoCleft software and ranked according to two criteria (number of atoms in common and Tanimoto score of local structural similarity) and the associated Z-score and p-value measures of statistical significance. The results, including predicted ligands, target proteins, similarity scores, number of atoms in common, etc., are shown in a powerful interactive graphical interface. This interface permits the visualization of target ligands superimposed on the query cavity and additionally provides a table of pairwise ligand topological similarities. Similarities between top scoring ligands serve as an additional tool to judge the quality of the results obtained. We present several examples where IsoCleft Finder provides useful functional information. IsoCleft Finder results are complementary to existing approaches for the prediction of protein function from structure, rational drug design and x-ray crystallography. IsoCleft Finder can be found at: http://bcb.med.usherbrooke.ca/isocleftfinder.
doi:10.12688/f1000research.2-117.v2
PMCID: PMC3892921  PMID: 24555058
4.  REFMAC5 for the refinement of macromolecular crystal structures 
The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described.
This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, ‘jelly-body’ restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback–Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.
doi:10.1107/S0907444911001314
PMCID: PMC3069751  PMID: 21460454
REFMAC5; refinement
5.  Structural Refinement from Restrained-Ensemble Simulations Based on EPR/DEER Data: Application to T4 Lysozyme 
The journal of physical chemistry. B  2013;117(17):4740-4754.
DEER (Double Electron Electron Resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parameterized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement.
doi:10.1021/jp311723a
PMCID: PMC3684008  PMID: 23510103
Structural refinement; spin-labeled T4 Lysozyme; ESR/DEER; rotamer population; molecular dynamics (MD); restrained ensemble (RE)
6.  Flexible torsion-angle noncrystallographic symmetry restraints for improved macromolecular structure refinement 
Flexible torsion angle-based NCS restraints have been implemented in phenix.refine, allowing improved model refinement at all resolutions. Rotamer correction and rotamer consistency checks between NCS-related amino-acid side chains further improve the final model quality.
One of the great challenges in refining macromolecular crystal structures is a low data-to-parameter ratio. Historically, knowledge from chemistry has been used to help to improve this ratio. When a macromolecule crystallizes with more than one copy in the asymmetric unit, the noncrystallographic symmetry relationships can be exploited to provide additional restraints when refining the working model. However, although globally similar, NCS-related chains often have local differences. To allow for local differences between NCS-related molecules, flexible torsion-based NCS restraints have been introduced, coupled with intelligent rotamer handling for protein chains, and are available in phenix.refine for refinement of models at all resolutions.
doi:10.1107/S1399004714003277
PMCID: PMC4014122  PMID: 24816103
macromolecular crystallography; noncrystallographic symmetry; NCS; refinement; automation
7.  “Conditional Restraints”: Restraining the Free Atoms in ARP/wARP 
Structure(London, England:1993)  2009;17(2-3):183-189.
Summary
The automated building of a protein model into an electron density map remains a challenging problem. In the ARP/wARP approach, model building is facilitated by initially interpreting a density map with free atoms of unknown chemical identity; all structural information for such chemically unassigned atoms is discarded. Here, this is remedied by applying restraints between free atoms, and between free atoms and a partial protein model. These are based on geometric considerations of protein structure and tentative (conditional) assignments for the free atoms. Restraints are applied in the REFMAC5 refinement program and are generated on an ad hoc basis, allowing them to fluctuate from step to step. A large set of experimentally phased and molecular replacement structures showcases individual structures where automated building is improved drastically by the conditional restraints. The concept and implementation we present can also find application in restraining geometries, such as hydrogen bonds, in low-resolution refinement.
doi:10.1016/j.str.2008.12.011
PMCID: PMC2670983  PMID: 19217389
PROTEINS; CELLBIO
8.  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
9.  A conformation-dependent stereochemical library improves crystallographic refinement even at atomic resolution 
A script was created to allow SHELXL to use the new CDL v.1.2 stereochemical library which defines the target values for main-chain bond lengths and angles as a function of the residue’s ϕ/ψ angles. Test refinements using this script show that the refinement behavior of structures at resolutions even better than 1 Å is substantially enhanced by the use of the new conformation-dependent ideal geometry paradigm.
To utilize a new conformation-dependent backbone-geometry library (CDL) in protein refinements at atomic resolution, a script was written that creates a restraint file for the SHELXL refinement program. It was found that the use of this library allows models to be created that have a substantially better fit to main-chain bond angles and lengths without degrading their fit to the X-ray data even at resolutions near 1 Å. For models at much higher resolution (∼0.7 Å), the refined model for parts adopting single well occupied positions is largely independent of the restraints used, but these structures still showed much smaller r.m.s.d. residuals when assessed with the CDL. Examination of the refinement tests across a wide resolution range from 2.4 to 0.65 Å revealed consistent behavior supporting the use of the CDL as a next-generation restraint library to improve refinement. CDL restraints can be generated using the service at http://pgd.science.oregonstate.edu/cdl_shelxl/.
doi:10.1107/S090744491102292X
PMCID: PMC3144852  PMID: 21795811
stereochemical libraries; refinement; conformation-dependent library
10.  Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures 
We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5 – 22 kDa, restrained-Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 38 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained-Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10 – 25 kDa the less cpu intensive restrained-Rosetta refinement protocols provided more accurate structures. The restrained-Rosetta protocols described here can improve the accuracy of protein NMR structures, and should find broad and general for studies of protein structure and function.
doi:10.1021/ja409845w
PMCID: PMC4129517  PMID: 24392845
11.  An introduction to stereochemical restraints 
A brief summary of the types of restraint defined in refinement dictionaries.
At the resolution available from most macromolecular crystals, the X-ray data alone are insufficient to lead to a chemically reasonable structure, so stereochemical restraints are essential. These usually restrain bond lengths, bond angles, planes and chiral volumes. The definition of these restraints and where the values come from are described. A dictionary entry contains information about the atom types, their connectivity and all the appropriate restraints. Torsion angles are not usually restrained, but they do have optimum values. In the special case of flexible five- and six-membered rings, including pentose and hexose sugars, the ring pucker is defined by combinations of torsion angles and the pucker affects the position of substituents.
doi:10.1107/S090744490604604X
PMCID: PMC2483478  PMID: 17164527
stereochemistry; restraints; bond lengths; bond angles; protein structure; crystallographic refinement
12.  Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum  
DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols.
Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
doi:10.1107/S090744491104978X
PMCID: PMC3322598  PMID: 22505259
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
13.  Rappertk: a versatile engine for discrete restraint-based conformational sampling of macromolecules 
Background
Macromolecular structures are modeled by conformational optimization within experimental and knowledge-based restraints. Discrete restraint-based sampling generates high-quality structures within these restraints and facilitates further refinement in a continuous all-atom energy landscape. This approach has been used successfully for protein loop modeling, comparative modeling and electron density fitting in X-ray crystallography.
Results
Here we present a software toolkit (Rappertk) which generalizes discrete restraint-based sampling for use in structural biology. Modular design and multi-layered architecture enables Rappertk to sample conformations of any macromolecule at many levels of detail and within a variety of experimental restraints. Performance against a Cα-tracing benchmark shows that the efficiency has not suffered despite the overhead required by this flexibility. We demonstrate the toolkit's capabilities by building high-quality β-sheets and by introducing restraint-driven sampling. RNA sampling is demonstrated by rebuilding a protein-RNA interface. Ability to construct arbitrary ligands is used in sampling protein-ligand interfaces within electron density. Finally, secondary structure and shape information derived from EM are combined to generate multiple conformations of a protein consistent with the observed density.
Conclusion
Through its modular design and ease of use, Rappertk enables exploration of a wide variety of interesting avenues in structural biology. This toolkit, with illustrative examples, is freely available to academic users from .
doi:10.1186/1472-6807-7-13
PMCID: PMC1847436  PMID: 17376228
14.  Conformation-independent structural comparison of macromolecules with ProSMART  
The Procrustes Structural Matching Alignment and Restraints Tool (ProSMART) has been developed to allow local comparative structural analyses independent of the global conformations and sequence homology of the compared macromolecules. This allows quick and intuitive visualization of the conservation of backbone and side-chain conformations, providing complementary information to existing methods.
The identification and exploration of (dis)similarities between macromolecular structures can help to gain biological insight, for instance when visualizing or quantifying the response of a protein to ligand binding. Obtaining a residue alignment between compared structures is often a prerequisite for such comparative analysis. If the conformational change of the protein is dramatic, conventional alignment methods may struggle to provide an intuitive solution for straightforward analysis. To make such analyses more accessible, the Procrustes Structural Matching Alignment and Restraints Tool (ProSMART) has been developed, which achieves a conformation-independent structural alignment, as well as providing such additional functionalities as the generation of restraints for use in the refinement of macromolecular models. Sensible comparison of protein (or DNA/RNA) structures in the presence of conformational changes is achieved by enforcing neither chain nor domain rigidity. The visualization of results is facilitated by popular molecular-graphics software such as CCP4mg and PyMOL, providing intuitive feedback regarding structural conservation and subtle dissimilarities between close homologues that can otherwise be hard to identify. Automatically generated colour schemes corresponding to various residue-based scores are provided, which allow the assessment of the conservation of backbone and side-chain conformations relative to the local coordinate frame. Structural comparison tools such as ProSMART can help to break the complexity that accompanies the constantly growing pool of structural data into a more readily accessible form, potentially offering biological insight or influencing subsequent experiments.
doi:10.1107/S1399004714016241
PMCID: PMC4157452  PMID: 25195761
ProSMART; Procrustes; structural comparison; alignment; external restraints; refinement
15.  Specific Non-Local Interactions Are Not Necessary for Recovering Native Protein Dynamics 
PLoS ONE  2014;9(3):e91347.
The elastic network model (ENM) is a widely used method to study native protein dynamics by normal mode analysis (NMA). In ENM we need information about all pairwise distances, and the distance between contacting atoms is restrained to the native value. Therefore ENM requires O(N2) information to realize its dynamics for a protein consisting of N amino acid residues. To see if (or to what extent) such a large amount of specific structural information is required to realize native protein dynamics, here we introduce a novel model based on only O(N) restraints. This model, named the ‘contact number diffusion’ model (CND), includes specific distance restraints for only local (along the amino acid sequence) atom pairs, and semi-specific non-local restraints imposed on each atom, rather than atom pairs. The semi-specific non-local restraints are defined in terms of the non-local contact numbers of atoms. The CND model exhibits the dynamic characteristics comparable to ENM and more correlated with the explicit-solvent molecular dynamics simulation than ENM. Moreover, unrealistic surface fluctuations often observed in ENM were suppressed in CND. On the other hand, in some ligand-bound structures CND showed larger fluctuations of buried protein atoms interacting with the ligand compared to ENM. In addition, fluctuations from CND and ENM show comparable correlations with the experimental B-factor. Although there are some indications of the importance of some specific non-local interactions, the semi-specific non-local interactions are mostly sufficient for reproducing the native protein dynamics.
doi:10.1371/journal.pone.0091347
PMCID: PMC3953337  PMID: 24625758
16.  Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling 
PLoS ONE  2013;8(6):e65770.
Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing the degrees of freedom. The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using -carbon positions only and sampling conformations with a Monte Carlo simulated annealing based protocol. Backbone atoms were added using a method previously described and then gradient minimised in the Rosetta force field. Despite the CG potential energy function being sequence-independent, the method performed similarly to methods that explicitly use either fragments of known protein backbones with similar sequences or residue-specific /-maps to restrict the search space. The method was also able to predict with sub-Angstrom accuracy two out of seven loops from recently solved crystal structures of proteins with low sequence and structure similarity to previously deposited structures in the PDB. The ability to sample realistic loop conformations directly from a potential energy function enables the incorporation of additional geometric restraints and the use of more advanced sampling methods in a way that is not possible to do easily with fragment replacement methods and also enable multi-scale simulations for protein design and protein structure prediction. These restraints could be derived from experimental data or could be design restraints in the case of computational protein design. C++ source code is available for download from http://www.sbg.bio.ic.ac.uk/phyre2/PD2/.
doi:10.1371/journal.pone.0065770
PMCID: PMC3688807  PMID: 23824634
17.  A Geometric Arrangement Algorithm for Structure Determination of Symmetric Protein Homo-Oligomers from NOEs and RDCs 
Journal of Computational Biology  2011;18(11):1507-1523.
Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a primary tool to perform structural studies of proteins in physiologically-relevant solution conditions. Restraints on distances between pairs of nuclei in the protein, derived from the nuclear Overhauser effect (NOE), provide information about the structure of the protein in its folded state. NMR studies of symmetric protein homo-oligomers present a unique challenge. Using X-filtered NOESY experiments, it is possible to determine whether an NOE restrains a pair of protons across different subunits or within a single subunit, but current experimental techniques are unable to determine in which subunits the restrained protons lie. Consequently, it is difficult to assign NOEs to particular pairs of subunits with certainty, thus hindering the structural analysis of the oligomeric state. Computational approaches are needed to address this subunit ambiguity, but traditional solutions often rely on stochastic search coupled with simulated annealing and simulations of simplified molecular dynamics, which have many tunable parameters that must be chosen carefully and can also fail to report structures consistent with the experimental restraints. In addition, these traditional approaches rarely provide guarantees on running time or solution quality. We reduce the structure determination of homo-oligomers with cyclic symmetry to computing geometric arrangements of unions of annuli in a plane. Our algorithm, disco, runs in expected O(n2) time, where n is the number of distance restraints, potentially assigned ambiguously. disco is guaranteed to report the exact set of oligomer structures consistent with the distance restraints and also with orientational restraints from residual dipolar couplings (RDCs). We demonstrate our method using two symmetric protein complexes: the trimeric E. coli diacylglycerol kinase (DAGK) and a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G (GB1). In both cases, disco computes oligomer structures with high precision and also finds distance restraints that are either mutually inconsistent or inconsistent with the RDCs. The entire protocol DISCO has been completely automated in a software package that is freely available and open-source at www.cs.duke.edu/donaldlab/software.php.
doi:10.1089/cmb.2011.0173
PMCID: PMC3216109  PMID: 22035328
algorithms; computational molecular biology; protein structure
18.  TagDock: An efficient rigid body docking algorithm for oligomeric protein complex model construction and experiment planning 
Biochemistry  2013;52(33):5577-5584.
We report here new computational tools and strategies to efficiently generate three-dimensional models for oligomeric biomolecular complexes in cases where there is limited experimental restraint data to guide the docking calculations. Our computational tools are designed to rapidly and exhaustively enumerate all geometrically possible docking poses for an oligomeric complex, rather than generate detailed, atomic-resolution models. Experimental data, such as inter-atomic distance measurements, are then used to select and refine docking poses that are consistent with the experimental restraints. Our computational toolkit is designed for use with sparse datasets to generate intermediate-resolution docking models, and utilizes distance difference matrix analysis to identify further restraint measurements that will provide maximum additional structural refinement. Thus, these tools can be used to help plan optimal residue positions for probe incorporation in labor-intensive biophysical experiments such as chemical crosslinking, EPR, or FRET spectroscopy studies. We present benchmark results for docking the collection of all 176 heterodimer protein complexes from the ZDOCK database, as well as a protein homodimer with recently collected experimental distance restraints, to illustrate the toolkit’s capabilities and performance, and to demonstrate how distance difference matrix analysis can automatically identify and prioritize additional restraint measurements that allow us to rapidly optimize docking poses.
doi:10.1021/bi400158k
PMCID: PMC3804560  PMID: 23875708
19.  Use of intensity quotients and differences in absolute structure refinement 
Differences and quotients can be defined using Friedel pairs of reflections and applied in refinement to enable absolute structure to be determined precisely even for light atom crystal structures.
Several methods for absolute structure refinement were tested using single-crystal X-ray diffraction data collected using Cu Kα radiation for 23 crystals with no element heavier than oxygen: conventional refinement using an inversion twin model, estimation using intensity quotients in SHELXL2012, estimation using Bayesian methods in PLATON, estimation using restraints consisting of numerical intensity differences in CRYSTALS and estimation using differences and quotients in TOPAS-Academic where both quantities were coded in terms of other structural parameters and implemented as restraints. The conventional refinement approach yielded accurate values of the Flack parameter, but with standard uncertainties ranging from 0.15 to 0.77. The other methods also yielded accurate values of the Flack parameter, but with much higher precision. Absolute structure was established in all cases, even for a hydrocarbon. The procedures in which restraints are coded explicitly in terms of other structural parameters enable the Flack parameter to correlate with these other parameters, so that it is determined along with those parameters during refinement.
doi:10.1107/S2052519213010014
PMCID: PMC3661305  PMID: 23719469
intensity quotients; absolute structure refinement
20.  Structure of the 1,N2-Etheno-2′-deoxyguanosine Adduct in Duplex DNA at pH 8.6† 
Chemical research in toxicology  2007;20(11):1601-1611.
The structure of the 1,N2-εdG adduct, arising from the reaction of vinyl chloride with dG, was determined in the oligonucleotide duplex 5′-d(CGCATXGAATCC)-3′•5′-d(GGATTCCATGCG)-3′ (X=1,N2-εdG) at pH 8.6 using high resolution NMR spectroscopy. The exocyclic lesion prevented Watson-Crick base-pairing capability at the adduct site and resulted in an ~17 °C of the C decrease in Tm oligodeoxynucleotide duplex. At neutral pH, conformational exchange resulted in spectral line broadening near the adducted site, and it was not possible to determine the structure. However, at pH 8.6, it was possible to obtain well-resolved 1H NMR spectra. This enabled a total of 385 NOE based distance restraints to be obtained, consisting of 245 intra- and 140 inter-nucleotide distances. The 31P NMR spectra exhibited two downfield-shifted resonances, suggesting a localized perturbation of the DNA backbone. The two downfield 31P resonances were assigned to G7 and C19. The solution structure was refined by molecular dynamics calculations restrained by NMR derived distance and dihedral angle restraints, using a simulated annealing protocol. The generalized Born approximation was used to simulate solvent. The emergent structures indicated that the 1,N2-εdG-induced structural perturbation was localized at the X6•C19 base pair, and its 5′-neighbor T5•A20. Both 1,N2-εdG and the complementary dC adopted the anti conformation about the glycosyl bonds. The 1,N2-εdG adduct was inserted into the duplex but was shifted towards the minor groove as compared to dG in a normal Watson-Crick C•G base pair. The complementary cytosine was displaced toward the major groove. The 5′-neighbor T5•A20 base pair was destabilized with respect to Watson-Crick base pairing. The refined structure predicted a bend in the helical axis associated with the adduct site.
doi:10.1021/tx7001788
PMCID: PMC2546360  PMID: 17941687
21.  A restraint molecular dynamics and simulated annealing approach for protein homology modeling utilizing mean angles 
BMC Bioinformatics  2005;6:91.
Background
We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Döker et al. (1999, BBRC, 257, 348–350). The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS.
Results
To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor γ (Ppar γ). The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar γ was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms.
Conclusion
In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data.
doi:10.1186/1471-2105-6-91
PMCID: PMC1127110  PMID: 15819976
22.  Understanding Uncertainties in Model-Based Predictions of Aedes aegypti Population Dynamics 
Background
Aedes aegypti is one of the most important mosquito vectors of human disease. The development of spatial models for Ae. aegypti provides a promising start toward model-guided vector control and risk assessment, but this will only be possible if models make reliable predictions. The reliability of model predictions is affected by specific sources of uncertainty in the model.
Methodology/Principal Findings
This study quantifies uncertainties in the predicted mosquito population dynamics at the community level (a cluster of 612 houses) and the individual-house level based on Skeeter Buster, a spatial model of Ae. aegypti, for the city of Iquitos, Peru. The study considers two types of uncertainty: 1) uncertainty in the estimates of 67 parameters that describe mosquito biology and life history, and 2) uncertainty due to environmental and demographic stochasticity. Our results show that for pupal density and for female adult density at the community level, respectively, the 95% prediction confidence interval ranges from 1000 to 3000 and from 700 to 5,000 individuals. The two parameters contributing most to the uncertainties in predicted population densities at both individual-house and community levels are the female adult survival rate and a coefficient determining weight loss due to energy used in metabolism at the larval stage (i.e. metabolic weight loss). Compared to parametric uncertainty, stochastic uncertainty is relatively low for population density predictions at the community level (less than 5% of the overall uncertainty) but is substantially higher for predictions at the individual-house level (larger than 40% of the overall uncertainty). Uncertainty in mosquito spatial dispersal has little effect on population density predictions at the community level but is important for the prediction of spatial clustering at the individual-house level.
Conclusion/Significance
This is the first systematic uncertainty analysis of a detailed Ae. aegypti population dynamics model and provides an approach for identifying those parameters for which more accurate estimates would improve model predictions.
Author Summary
Dengue is one of the most important insect-vectored human viral diseases. The principal vector is Aedes aegypti, a mosquito that lives in close association with humans. Currently, there is no effective vaccine available and the only means for limiting dengue outbreaks is vector control. To help design vector control strategies, spatial models of Ae. aegypti population dynamics have been developed. However, the usefulness of such models depends on the reliability of their predictions, which can be affected by different sources of uncertainty including uncertainty in the model parameter estimation, uncertainty in the model structure, measurement errors in the data fed into the model, individual variability, and stochasticity in the environment. This study quantifies uncertainties in the mosquito population dynamics predicted by Skeeter Buster, a spatial model of Ae. aegypti, for the city of Iquitos, Peru. The uncertainty quantification should enable us to better understand the reliability of model predictions, improve Skeeter Buster and other similar models by targeting those parameters with high uncertainty contributions for further empirical research, and thereby decrease uncertainty in model predictions.
doi:10.1371/journal.pntd.0000830
PMCID: PMC2946899  PMID: 20927187
23.  Low-resolution refinement tools in REFMAC5 
Low-resolution refinement tools implemented in REFMAC5 are described, including the use of external structural restraints, helical restraints and regularized anisotropic map sharpening.
Two aspects of low-resolution macromolecular crystal structure analysis are considered: (i) the use of reference structures and structural units for provision of structural prior information and (ii) map sharpening in the presence of noise and the effects of Fourier series termination. The generation of interatomic distance restraints by ProSMART and their subsequent application in REFMAC5 is described. It is shown that the use of such external structural information can enhance the reliability of derived atomic models and stabilize refinement. The problem of map sharpening is considered as an inverse deblurring problem and is solved using Tikhonov regularizers. It is demonstrated that this type of map sharpening can automatically produce a map with more structural features whilst maintaining connectivity. Tests show that both of these directions are promising, although more work needs to be performed in order to further exploit structural information and to address the problem of reliable electron-density calculation.
doi:10.1107/S090744491105606X
PMCID: PMC3322599  PMID: 22505260
low-resolution refinement; REFMAC5
24.  PICKY: a novel SVD-based NMR spectra peak picking method 
Bioinformatics  2009;25(12):i268-i275.
Motivation: Picking peaks from experimental NMR spectra is a key unsolved problem for automated NMR protein structure determination. Such a process is a prerequisite for resonance assignment, nuclear overhauser enhancement (NOE) distance restraint assignment, and structure calculation tasks. Manual or semi-automatic peak picking, which is currently the prominent way used in NMR labs, is tedious, time consuming and costly.
Results: We introduce new ideas, including noise-level estimation, component forming and sub-division, singular value decomposition (SVD)-based peak picking and peak pruning and refinement. PICKY is developed as an automated peak picking method. Different from the previous research on peak picking, we provide a systematic study of the proposed method. PICKY is tested on 32 real 2D and 3D spectra of eight target proteins, and achieves an average of 88% recall and 74% precision. PICKY is efficient. It takes PICKY on average 15.7 s to process an NMR spectrum. More important than these numbers, PICKY actually works in practice. We feed peak lists generated by PICKY to IPASS for resonance assignment, feed IPASS assignment to SPARTA for fragments generation, and feed SPARTA fragments to FALCON for structure calculation. This results in high-resolution structures of several proteins, for example, TM1112, at 1.25 Å.
Availability: PICKY is available upon request. The peak lists of PICKY can be easily loaded by SPARKY to enable a better interactive strategy for rapid peak picking.
Contact: mli@uwaterloo.ca
doi:10.1093/bioinformatics/btp225
PMCID: PMC2687979  PMID: 19477998
25.  PDB_REDO: constructive validation, more than just looking for errors 
The decision-making algorithms and software used in PDB_REDO to re-refine and rebuild crystallographic protein structures in the PDB are presented and discussed.
Developments of the PDB_REDO procedure that combine re-refinement and rebuilding within a unique decision-making framework to improve structures in the PDB are presented. PDB_REDO uses a variety of existing and custom-built software modules to choose an optimal refinement protocol (e.g. anisotropic, isotropic or overall B-factor refinement, TLS model) and to optimize the geometry versus data-refinement weights. Next, it proceeds to rebuild side chains and peptide planes before a final optimization round. PDB_REDO works fully automatically without the need for intervention by a crystallographic expert. The pipeline was tested on 12 000 PDB entries and the great majority of the test cases improved both in terms of crystallographic criteria such as R free and in terms of widely accepted geometric validation criteria. It is concluded that PDB_REDO is useful to update the otherwise ‘static’ structures in the PDB to modern crystallographic standards. The publically available PDB_REDO database provides better model statistics and contributes to better refinement and validation targets.
doi:10.1107/S0907444911054515
PMCID: PMC3322608  PMID: 22505269
validation; refinement; model building; automation; PDB

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