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1.  Model morphing and sequence assignment after molecular replacement 
A procedure for model building is described that combines morphing a model to match a density map, trimming the morphed model and aligning the model to a sequence.
A procedure termed ‘morphing’ for improving a model after it has been placed in the crystallographic cell by molecular replacement has recently been developed. Morphing consists of applying a smooth deformation to a model to make it match an electron-density map more closely. Morphing does not change the identities of the residues in the chain, only their coordinates. Consequently, if the true structure differs from the working model by containing different residues, these differences cannot be corrected by morphing. Here, a procedure that helps to address this limitation is described. The goal of the procedure is to obtain a relatively complete model that has accurate main-chain atomic positions and residues that are correctly assigned to the sequence. Residues in a morphed model that do not match the electron-density map are removed. Each segment of the resulting trimmed morphed model is then assigned to the sequence of the molecule using information about the connectivity of the chains from the working model and from connections that can be identified from the electron-density map. The procedure was tested by application to a recently determined structure at a resolution of 3.2 Å and was found to increase the number of correctly identified residues in this structure from the 88 obtained using phenix.resolve sequence assignment alone (Terwilliger, 2003 ▶) to 247 of a possible 359. Additionally, the procedure was tested by application to a series of templates with sequence identities to a target structure ranging between 7 and 36%. The mean fraction of correctly identified residues in these cases was increased from 33% using phenix.resolve sequence assignment to 47% using the current procedure. The procedure is simple to apply and is available in the Phenix software package.
PMCID: PMC3817698  PMID: 24189236
morphing; model building; sequence assignment; model–map correlation; loop-building
2.  The JCSG MR pipeline: optimized alignments, multiple models and parallel searches 
The practical limits of molecular replacement can be extended by using several specifically designed protein models based on fold-recognition methods and by exhaustive searches performed in a parallelized pipeline. Updated results from the JCSG MR pipeline, which to date has solved 33 molecular-replacement structures with less than 35% sequence identity to the closest homologue of known structure, are presented.
The success rate of molecular replacement (MR) falls considerably when search models share less than 35% sequence identity with their templates, but can be improved significantly by using fold-recognition methods combined with exhaustive MR searches. Models based on alignments calculated with fold-recognition algorithms are more accurate than models based on conventional alignment methods such as FASTA or BLAST, which are still widely used for MR. In addition, by designing MR pipelines that integrate phasing and automated refinement and allow parallel processing of such calculations, one can effectively increase the success rate of MR. Here, updated results from the JCSG MR pipeline are presented, which to date has solved 33 MR structures with less than 35% sequence identity to the closest homologue of known structure. By using difficult MR problems as examples, it is demonstrated that successful MR phasing is possible even in cases where the similarity between the model and the template can only be detected with fold-recognition algorithms. In the first step, several search models are built based on all homologues found in the PDB by fold-recognition algorithms. The models resulting from this process are used in parallel MR searches with different combinations of input parameters of the MR phasing algorithm. The putative solutions are subjected to rigid-body and restrained crystallo­graphic refinement and ranked based on the final values of free R factor, figure of merit and deviations from ideal geometry. Finally, crystal packing and electron-density maps are checked to identify the correct solution. If this procedure does not yield a solution with interpretable electron-density maps, then even more alternative models are prepared. The structurally variable regions of a protein family are identified based on alignments of sequences and known structures from that family and appropriate trimmings of the models are proposed. All combinations of these trimmings are applied to the search models and the resulting set of models is used in the MR pipeline. It is estimated that with the improvements in model building and exhaustive parallel searches with existing phasing algorithms, MR can be successful for more than 50% of recognizable homologues of known structures below the threshold of 35% sequence identity. This implies that about one-third of the proteins in a typical bacterial proteome are potential MR targets.
PMCID: PMC2394805  PMID: 18094477
molecular replacement; sequence-alignment accuracy; homology modeling; parameter-space screening; structural genomics
3.  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.
PMCID: PMC3322598  PMID: 22505259
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
4.  Statistical density modification using local pattern matching 
Statistical density modification can make use of local patterns of density found in protein structures to improve crystallographic phases.
A method for improving crystallographic phases is presented that is based on the preferential occurrence of certain local patterns of electron density in macromolecular electron-density maps. The method focuses on the relationship between the value of electron density at a point in the map and the pattern of density surrounding this point. Patterns of density that can be superimposed by rotation about the central point are considered equivalent. Standard templates are created from experimental or model electron-density maps by clustering and averaging local patterns of electron density. The clustering is based on correlation coefficients after rotation to maximize the correlation. Experimental or model maps are also used to create histograms relating the value of electron density at the central point to the correlation coefficient of the density surrounding this point with each member of the set of standard patterns. These histograms are then used to estimate the electron density at each point in a new experimental electron-density map using the pattern of electron density at points surrounding that point and the correlation coefficient of this density to each of the set of standard templates, again after rotation to maximize the correlation. The method is strengthened by excluding any information from the point in question from both the templates and the local pattern of density in the calculation. A function based on the origin of the Patterson function is used to remove information about the electron density at the point in question from nearby electron density. This allows an estimation of the electron density at each point in a map, using only information from other points in the process. The resulting estimates of electron density are shown to have errors that are nearly independent of the errors in the original map using model data and templates calculated at a resolution of 2.6 Å. Owing to this independence of errors, information from the new map can be combined in a simple fashion with information from the original map to create an improved map. An iterative phase-improvement process using this approach and other applications of the image-reconstruction method are described and applied to experimental data at resolutions ranging from 2.4 to 2.8 Å.
PMCID: PMC2745877  PMID: 14501107
density modification; pattern matching
5.  Improving macromolecular atomic models at moderate resolution by automated iterative model building, statistical density modification and refinement 
A procedure for iterative model-building, statistical density modification and refinement at moderate resolution (up to about 2.8 Å) is described.
An iterative process for improving the completeness and quality of atomic models automatically built at moderate resolution (up to about 2.8 Å) is described. The process consists of cycles of model building interspersed with cycles of refinement and combining phase information from the model with experimental phase information (if any) using statistical density modification. The process can lead to substantial improvements in both the accuracy and completeness of the model compared with a single cycle of model building. For eight test cases solved by MAD or SAD at resolutions ranging from 2.0 to 2.8 Å, the fraction of models built and assigned to sequence was 46–91% (mean of 65%) after the first cycle of building and refinement, and 78–­95% (mean of 87%) after 20 cycles. In an additional test case, an incorrect model of gene 5 protein (PDB code 2gn5; r.m.s.d. of main-chain atoms from the more recent refined structure 1vqb at 1.56 Å) was rebuilt using only structure-factor amplitude information at varying resolutions from 2.0 to 3.0 Å. Rebuilding was effective at resolutions up to about 2.5 Å. The resulting models had 60–­80% of the residues built and an r.m.s.d. of main-chain atoms from the refined structure of 0.20 to 0.62 Å. The algorithm is useful for building preliminary models of macromolecules suitable for an experienced crystallographer to extend, correct and fully refine.
PMCID: PMC2745880  PMID: 12832760
density modification; model building; refinement
6.  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
PMCID: PMC3036769  PMID: 20850544
Ligand-binding site refinement; proteinthreading; protein structure prediction; ligand-binding site prediction; ensemble docking; molecular function
7.  The PDB_REDO server for macromolecular structure model optimization 
IUCrJ  2014;1(Pt 4):213-220.
The PDB_REDO pipeline aims to improve macromolecular structures by optimizing the crystallographic refinement parameters and performing partial model building. Here, algorithms are presented that allowed a web-server implementation of PDB_REDO, and the first user results are discussed.
The refinement and validation of a crystallographic structure model is the last step before the coordinates and the associated data are submitted to the Protein Data Bank (PDB). The success of the refinement procedure is typically assessed by validating the models against geometrical criteria and the diffraction data, and is an important step in ensuring the quality of the PDB public archive [Read et al. (2011 ▶), Structure, 19, 1395–1412]. The PDB_REDO procedure aims for ‘constructive validation’, aspiring to consistent and optimal refinement parameterization and pro-active model rebuilding, not only correcting errors but striving for optimal interpretation of the electron density. A web server for PDB_REDO has been implemented, allowing thorough, consistent and fully automated optimization of the refinement procedure in REFMAC and partial model rebuilding. The goal of the web server is to help practicing crystallo­graphers to improve their model prior to submission to the PDB. For this, additional steps were implemented in the PDB_REDO pipeline, both in the refinement procedure, e.g. testing of resolution limits and k-fold cross-validation for small test sets, and as new validation criteria, e.g. the density-fit metrics implemented in EDSTATS and ligand validation as implemented in YASARA. Innovative ways to present the refinement and validation results to the user are also described, which together with auto-generated Coot scripts can guide users to subsequent model inspection and improvement. It is demonstrated that using the server can lead to substantial improvement of structure models before they are submitted to the PDB.
PMCID: PMC4107921  PMID: 25075342
PDB_REDO; validation; model optimization
8.  Building and Refining Protein Models within Cryo-electron Microscopy Density Maps Based on Homology Modeling and Multi-scale Structure Refinement 
Journal of molecular biology  2010;397(3):835-851.
Automatic modeling methods using cryo-electron microscopy (cryoEM) density maps as constrains are promising approaches to building atomic models of individual proteins or protein domains. However, their application to large macromolecular assemblies has not been possible largely due to computational limitations inherent to such unsupervised methods. Here we describe a new method, EM-IMO, for building, modifying and refining local structures of protein models using cryoEM maps as a constraint. As a supervised refinement method, EM-IMO allows users to specify parameters derived from inspections, so as to guide, and as a consequence, significantly speed up the refinement. An EM-IMO-based refinement protocol is first benchmarked on a data set of 50 homology models using simulated density maps. A multi-scale refinement strategy that combines EM-IMO-based and molecular dynamics (MD)-based refinement is then applied to build backbone models for the seven conformers of the five capsid proteins in our near-atomic resolution cryoEM map of the grass carp reovirus (GCRV) virion, a member of the aquareovirus genus of the Reoviridae family. The refined models allow us to reconstruct a backbone model of the entire GCRV capsid and provide valuable functional insights that are described in the accompanying publication. Our study demonstrates that the integrated use of homology modeling and a multi-scale refinement protocol that combines supervised and automated structure refinement offers a practical strategy for building atomic models based on medium- to high-resolution cryoEM density maps.
PMCID: PMC2860449  PMID: 20109465
cryo-electron microscopy; density fitting; homology modeling; structure refinement; protein structure prediction
9.  Ab initio protein modeling into cryoEM density maps using EM-Fold 
Biopolymers  2012;97(9):669-677.
EM-Fold was used to build models for nine proteins in the maps of GroEL (7.7 Å resolution) and ribosome (6.4 Å resolution) in the ab initio modeling category of the 2010 cryoEM modeling challenge. EM-Fold assembles predicted secondary structure elements (SSEs) into regions of the density map that were identified to correspond to either α-helices or β-strands. The assembly uses a Monte Carlo algorithm where loop closure, density-SSE length agreement, and strength of connecting density between SSEs are evaluated. Top scoring models are refined by translating, rotating and bending SSEs to yield better agreement with the density map. EM-Fold produces models that contain backbone atoms within secondary structure elements only. The RMSD values of the models with respect to native range from 2.4 Å to 3.5 Å for six of the nine proteins. EM-Fold failed to predict the correct topology in three cases. Subsequently Rosetta was used to build loops and side chains for the very best scoring models after EM-Fold refinement. The refinement within Rosetta’s force field is driven by a density agreement score that calculates a cross correlation between a density map simulated from the model and the experimental density map. All-atom RMSDs as low as 3.4 Å are achieved in favorable cases. Values above 10.0 Å are observed for two proteins with low overall content of secondary structure and hence particularly complex loop modeling problems. RMSDs over residues in secondary structure elements range from 2.5 Å to 4.8 Å.
PMCID: PMC3375387  PMID: 22302372
10.  Automated side-chain model building and sequence assignment by template matching 
A method for automated macromolecular side-chain model building and for aligning the sequence to the map is described.
An algorithm is described for automated building of side chains in an electron-density map once a main-chain model is built and for alignment of the protein sequence to the map. The procedure is based on a comparison of electron density at the expected side-chain positions with electron-density templates. The templates are constructed from average amino-acid side-chain densities in 574 refined protein structures. For each contiguous segment of main chain, a matrix with entries corresponding to an estimate of the probability that each of the 20 amino acids is located at each position of the main-chain model is obtained. The probability that this segment corresponds to each possible alignment with the sequence of the protein is estimated using a Bayesian approach and high-confidence matches are kept. Once side-chain identities are determined, the most probable rotamer for each side chain is built into the model. The automated procedure has been implemented in the RESOLVE software. Combined with automated main-chain model building, the procedure produces a preliminary model suitable for refinement and extension by an experienced crystallographer.
PMCID: PMC2745879  PMID: 12499538
model building; template matching
11.  Toward better refinement of comparative models: Predicting loops in inexact environments 
Proteins  2008;72(3):959-971.
Achieving atomic-level accuracy in comparative protein models is limited by our ability to refine the initial, homolog-derived model closer to the native state. Despite considerable effort, progress in developing a generalized refinement method has been limited. In contrast, methods have been described that can accurately reconstruct loop conformations in native protein structures. We hypothesize that loop refinement in homology models is much more difficult than loop reconstruction in crystal structures, in part, because side-chain, backbone, and other structural inaccuracies surrounding the loop create a challenging sampling problem; the loop cannot be refined without simultaneously refining adjacent portions. In this work, we single out one sampling issue in an artificial but useful test set and examine how loop refinement accuracy is affected by errors in surrounding side-chains. In 80 high-resolution crystal structures, we first perturbed 6–12 residue loops away from the crystal conformation, and placed all protein side chains in non-native but low energy conformations. Even these relatively small perturbations in the surroundings made the loop prediction problem much more challenging. Using a previously published loop prediction method, median backbone (N-Cα-CO) RMSD’s for groups of 6, 8, 10, and 12 residue loops are 0.3/0.6/0.4/0.6 Å, respectively, on native structures and increase to 1.1/2.2/1.5/2.3 Å on the perturbed cases. We then augmented our previous loop prediction method to simultaneously optimize the rotamer states of side chains surrounding the loop. Our results show that this augmented loop prediction method can recover the native state in many perturbed structures where the previous method failed; the median RMSD’s for the 6, 8, 10, and 12 residue perturbed loops improve to 0.4/0.8/1.1/1.2 Å. Finally, we highlight three comparative models from blind tests, in which our new method predicted loops closer to the native conformation than first modeled using the homolog template, a task generally understood to be difficult. Although many challenges remain in refining full comparative models to high accuracy, this work offers a methodical step toward that goal.
PMCID: PMC2764870  PMID: 18300241
comparative; homology; modeling; refinement; loop prediction; molecular mechanics; force field
12.  Distance Matrix-Based Approach to Protein Structure Prediction 
Much structural information is encoded in the internal distances; a distance matrix-based approach can be used to predict protein structure and dynamics, and for structural refinement. Our approach is based on the square distance matrix D = [rij2] containing all square distances between residues in proteins. This distance matrix contains more information than the contact matrix C, that has elements of either 0 or 1 depending on whether the distance rij is greater or less than a cutoff value rcutoff .We have performed spectral decomposition of the distance matrices D=∑λkVkVkT , in terms of eigenvalues λk and the corresponding eigenvectors vk and found that it contains at most 5 nonzero terms. A dominant eigenvector is proportional to r2 - the square distance of points from the center of mass, with the next three being the principal components of the system of points. By knowing r2 we can approximate a distance matrix of a protein with an expected RMSD value of about 4.5Å. We can also explain the role of hydrophobic interactions for the protein structure, because r is highly correlated with the hydrophobic profile of the sequence. Moreover, r is highly correlated with several sequence profiles which are useful in protein structure prediction, such as contact number, the residue-wise contact order (RWCO) or mean square fluctuations (i.e. crystallographic temperature factors). We have also shown that the next three components are related to spatial directionality of the secondary structure elements, and they may be also predicted from the sequence, improving overall structure prediction. We have also shown that the large number of available HIV-1 protease structures provides a remarkable sampling of conformations, which can be viewed as direct structural information about the dynamics. After structure matching, we apply principal component analysis (PCA) to obtain the important apparent motions for both bound and unbound structures. There are significant similarities between the first few key motions and the first few low-frequency normal modes calculated from a static representative structure with an elastic network model (ENM) that is based on the contact matrix C (related to D), strongly suggesting that the variations among the observed structures and the corresponding conformational changes are facilitated by the low-frequency, global motions intrinsic to the structure. Similarities are also found when the approach is applied to an NMR ensemble, as well as to atomic molecular dynamics (MD) trajectories. Thus, a sufficiently large number of experimental structures can directly provide important information about protein dynamics, but ENM can also provide a similar sampling of conformations. Finally, we use distance constraints from databases of known protein structures for structure refinement. We use the distributions of distances of various types in known protein structures to obtain the most probable ranges or the mean-force potentials for the distances. We then impose these constraints on structures to be refined or include the mean-force potentials directly in the energy minimization so that more plausible structural models can be built. This approach has been successfully used by us in 2006 in the CASPR structure refinement (
PMCID: PMC3018873  PMID: 19224393
13.  Automated 3D Motion Tracking using Gabor Filter Bank, Robust Point Matching, and Deformable Models 
Tagged Magnetic Resonance Imaging (tagged MRI or tMRI) provides a means of directly and noninvasively displaying the internal motion of the myocardium. Reconstruction of the motion field is needed to quantify important clinical information, e.g., the myocardial strain, and detect regional heart functional loss. In this paper, we present a three-step method for this task. First, we use a Gabor filter bank to detect and locate tag intersections in the image frames, based on local phase analysis. Next, we use an improved version of the Robust Point Matching (RPM) method to sparsely track the motion of the myocardium, by establishing a transformation function and a one-to-one correspondence between grid tag intersections in different image frames. In particular, the RPM helps to minimize the impact on the motion tracking result of: 1) through-plane motion, and 2) relatively large deformation and/or relatively small tag spacing. In the final step, a meshless deformable model is initialized using the transformation function computed by RPM. The model refines the motion tracking and generates a dense displacement map, by deforming under the influence of image information, and is constrained by the displacement magnitude to retain its geometric structure. The 2D displacement maps in short and long axis image planes can be combined to drive a 3D deformable model, using the Moving Least Square method, constrained by the minimization of the residual error at tag intersections. The method has been tested on a numerical phantom, as well as on in vivo heart data from normal volunteers and heart disease patients. The experimental results show that the new method has a good performance on both synthetic and real data. Furthermore, the method has been used in an initial clinical study to assess the differences in myocardial strain distributions between heart disease (left ventricular hypertrophy) patients and the normal control group. The final results show that the proposed method is capable of separating patients from healthy individuals. In addition, the method detects and makes possible quantification of local abnormalities in the myocardium strain distribution, which is critical for quantitative analysis of patients’ clinical conditions. This motion tracking approach can improve the throughput and reliability of quantitative strain analysis of heart disease patients, and has the potential for further clinical applications.
PMCID: PMC3742336  PMID: 19369149
Tagged MRI; Motion Tracking; Gabor Filter; RPM; Deformable Model; Strain
14.  A Generalized Learning Based Framework for Fast Brain Image Registration 
This paper presents a generalized learning based framework for improving both speed and accuracy of the existing deformable registration method. The key of our framework involves the utilization of a support vector regression (SVR) to learn the correlation between brain image appearances and their corresponding shape deformations to a template, for helping significantly cut down the computation cost and improve the robustness to local minima by using the learned correlation to instantly predict a good subject-specific deformation initialization for any given subject under registration. Our framework consists of three major parts: 1) training of SVR models based on the statistics of image samples and their shape deformations to capture intrinsic image-deformation correlations, 2) deformation prediction for a new subject with the trained SVR models to generate a subject-resemblance intermediate template by warping the original template with the predicted deformations, and 3) estimating of the residual deformation from the intermediate template to the subject for refined registration. Any existing deformable registration methods can be easily employed for training the SVR models and estimating the residual deformation. We have tested in this paper the two widely used deformable registration algorithms, i.e., HAMMER [1] and diffeomorphic demons [2], for demonstration of our proposed frameowrk. Experimental results show that, compared to the registration using the original methods (with no deformation prediction), our framework achieves a significant speedup (6X faster than HAMMER, and 3X faster than diffeomorphic demons), while maintaining comparable (or even slighly better) registration accuracy.
PMCID: PMC3021962  PMID: 20879329
15.  Super-resolution biomolecular crystallography with low-resolution data 
Nature  2010;464(7292):1218-1222.
X-ray diffraction plays a pivotal role in understanding of biological systems by revealing atomic structures of proteins, nucleic acids, and their complexes, with much recent interest in very large assemblies like the ribosome. Since crystals of such large assemblies often diffract weakly (resolution worse than 4 Å), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, while others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex1. Determining such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution below 5 Å generally exceed the number of degrees of freedom. Here we introduce a new method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with Rfree determines the optimum deformation and influence of the homology model. For test cases at 3.5 – 5 Å resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model coordinate accuracy, the definition of secondary structure, and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the PDB, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to studying weakly diffracting crystals using X-ray micro-diffraction2 as well as data from new X-ray light sources3. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to sub-nanometer resolution4,5, it can use similar tools.
PMCID: PMC2859093  PMID: 20376006
X-ray crystallography; homology modeling; cross-validation; Rfree value; refinement
16.  MAIN software for density averaging, model building, structure refinement and validation 
MAIN is interactive software designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. The features of MAIN and its tools for electron-density map calculations, model building, refinement in real and reciprocal space, and validation exploiting noncrystallographic symmetry in single and multiple crystal forms are presented.
MAIN is software that has been designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. Using MAIN, it is possible to perform density modification, manual and semi-automated or automated model building and rebuilding, real- and reciprocal-space structure optimization and refinement, map calculations and various types of molecular structure validation. The prompt availability of various analytical tools and the immediate visualization of molecular and map objects allow a user to efficiently progress towards the completed refined structure. The extraordinary depth perception of molecular objects in three dimensions that is provided by MAIN is achieved by the clarity and contrast of colours and the smooth rotation of the displayed objects. MAIN allows simultaneous work on several molecular models and various crystal forms. The strength of MAIN lies in its manipulation of averaged density maps and molecular models when noncrystallographic symmetry (NCS) is present. Using MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.
PMCID: PMC3727325  PMID: 23897458
molecular modelling; molecular graphics; macromolecular crystal structure determination; map calculation; computer programs
17.  Automated main-chain model building by template matching and iterative fragment extension 
A method for automated macromolecular main-chain model building is described.
An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and β-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and β-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more Cα positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 Å. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition.
PMCID: PMC2745878  PMID: 12499537
model building; template matching; fragment extension
18.  CaspR: a web server for automated molecular replacement using homology modelling 
Nucleic Acids Research  2004;32(Web Server issue):W606-W609.
Molecular replacement (MR) is the method of choice for X-ray crystallography structure determination when structural homologues are available in the Protein Data Bank (PDB). Although the success rate of MR decreases sharply when the sequence similarity between template and target proteins drops below 35% identical residues, it has been found that screening for MR solutions with a large number of different homology models may still produce a suitable solution where the original template failed. Here we present the web tool CaspR, implementing such a strategy in an automated manner. On input of experimental diffraction data, of the corresponding target sequence and of one or several potential templates, CaspR executes an optimized molecular replacement procedure using a combination of well-established stand-alone software tools. The protocol of model building and screening begins with the generation of multiple structure–sequence alignments produced with T-COFFEE, followed by homology model building using MODELLER, molecular replacement with AMoRe and model refinement based on CNS. As a result, CaspR provides a progress report in the form of hierarchically organized summary sheets that describe the different stages of the computation with an increasing level of detail. For the 10 highest-scoring potential solutions, pre-refined structures are made available for download in PDB format. Results already obtained with CaspR and reported on the web server suggest that such a strategy significantly increases the fraction of protein structures which may be solved by MR. Moreover, even in situations where standard MR yields a solution, pre-refined homology models produced by CaspR significantly reduce the time-consuming refinement process. We expect this automated procedure to have a significant impact on the throughput of large-scale structural genomics projects. CaspR is freely available at
PMCID: PMC441538  PMID: 15215460
19.  FoldGPCR: structure prediction protocol for the transmembrane domain of G protein-coupled receptors from class A 
Proteins  2010;78(10):2189-2201.
Building reliable structural models of G protein-coupled receptors (GPCRs) is a difficult task due to the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template-based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1–3 Å Cα RMSD of the native structure remains a major challenge. Here we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter-residue contacts between the template and target, and exploits an all-atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor-ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human β2-adrenergic receptor (β2AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison to the X-ray crystal structure of the β2AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix-helix packing interactions that distinguish rhodopsin from β2AR.
PMCID: PMC2933064  PMID: 20544957
class A GPCR; structure prediction; simulated annealing; ligand binding; implicit solvent; membrane protein
20.  Structure Refinement of Protein Low Resolution Models Using the GNEIMO Constrained Dynamics Method 
The Journal of Physical Chemistry. B  2012;116(8):2365-2375.
The challenge in protein structure prediction using homology modeling is the lack of reliable methods to refine the low resolution homology models. Unconstrained all-atom molecular dynamics (MD) does not serve well for structure refinement due to its limited conformational search. We have developed and tested the constrained MD method, based on the Generalized Newton-Euler Inverse Mass Operator (GNEIMO) algorithm for protein structure refinement. In this method, the high-frequency degrees of freedom are replaced with hard holonomic constraints and a protein is modeled as a collection of rigid body clusters connected by flexible torsional hinges. This allows larger integration time steps and enhances the conformational search space. In this work, we have demonstrated the use of a constraint free GNEIMO method for protein structure refinement that starts from low-resolution decoy sets derived from homology methods. In the eight proteins with three decoys for each, we observed an improvement of ~2 Å in the RMSD to the known experimental structures of these proteins. The GNEIMO method also showed enrichment in the population density of native-like conformations. In addition, we demonstrated structural refinement using a “Freeze and Thaw” clustering scheme with the GNEIMO framework as a viable tool for enhancing localized conformational search. We have derived a robust protocol based on the GNEIMO replica exchange method for protein structure refinement that can be readily extended to other proteins and possibly applicable for high throughput protein structure refinement.
PMCID: PMC3377353  PMID: 22260550
Constrained MD; GNEIMO; Structure Refinement; Decoys
21.  Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias 
An OMIT procedure is presented that has the benefits of iterative model building density modification and refinement yet is essentially unbiased by the atomic model that is built.
A procedure for carrying out iterative model building, density modification and refinement is presented in which the density in an OMIT region is essentially unbiased by an atomic model. Density from a set of overlapping OMIT regions can be combined to create a composite ‘iterative-build’ OMIT map that is everywhere unbiased by an atomic model but also everywhere benefiting from the model-based information present elsewhere in the unit cell. The procedure may have applications in the validation of specific features in atomic models as well as in overall model validation. The procedure is demonstrated with a molecular-replacement structure and with an experimentally phased structure and a variation on the method is demonstrated by removing model bias from a structure from the Protein Data Bank.
PMCID: PMC2424225  PMID: 18453687
model building; model validation; macromolecular models; Protein Data Bank; refinement; OMIT maps; bias; structure refinement; PHENIX
22.  GalaxyRefine: protein structure refinement driven by side-chain repacking 
Nucleic Acids Research  2013;41(Web Server issue):W384-W388.
The quality of model structures generated by contemporary protein structure prediction methods strongly depends on the degree of similarity between the target and available template structures. Therefore, the importance of improving template-based model structures beyond the accuracy available from template information has been emphasized in the structure prediction community. The GalaxyRefine web server, freely available at, is based on a refinement method that has been successfully tested in CASP10. The method first rebuilds side chains and performs side-chain repacking and subsequent overall structure relaxation by molecular dynamics simulation. According to the CASP10 assessment, this method showed the best performance in improving the local structure quality. The method can improve both global and local structure quality on average, when used for refining the models generated by state-of-the-art protein structure prediction servers.
PMCID: PMC3692086  PMID: 23737448
23.  High-Resolution Electron Microscopy of Helical Specimens: A Fresh Look at Tobacco Mosaic Virus 
Journal of molecular biology  2007;371(3):812-835.
The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be applied to a wide range of helical particles such as viruses, microtubules and helical filaments. We have made improvements to this approach using Tobacco Mosaic Virus (TMV) as a test specimen and obtained a map from 210,000 asymmetric units at a resolution better than 5 Å. The major improvements include the determination and correction of the contrast transfer function of the microscope. Alignment of helical segments was helped by constraints derived from the helical symmetry of the virus. Furthermore, symmetrization was implemented by multiple inclusions of symmetry-related views in the 3D reconstruction. We used the density map to build an atomic model of TMV. The model was refined using a real-space refinement strategy that accommodates multiple conformers. The atomic model shows significant deviations from the deposited model for the helical form of TMV at the lower-radius region (residues 88 to 109). This region appears more ordered with well-defined secondary structure, compared with the earlier helical structure. The RNA phosphate backbone is sandwiched between two arginine side chains, stabilizing the interaction between RNA and coat protein. A cluster of two or three carboxylates is buried in a hydrophobic environment isolating it from neighboring subunits. These carboxylates may represent the so-called Caspar carboxylates that form a metastable switch for viral disassembly. Overall, the observed differences suggest that the new model represents a different, more stable state of the virus, compared with the earlier published model.
PMCID: PMC2025690  PMID: 17585939
high-resolution single-particle electron microscopy; helical reconstruction; refinement; Tobacco Mosaic Virus; viral assembly/disassembly
24.  Advances in Rosetta structure prediction for difficult molecular-replacement problems 
Modeling advances using Rosetta structure prediction to aid in solving difficult molecular-replacement problems are discussed.
Recent work has shown the effectiveness of structure-prediction methods in solving difficult molecular-replacement problems. The Rosetta protein structure modeling suite can aid in the solution of difficult molecular-replacement problems using templates from 15 to 25% sequence identity; Rosetta refinement guided by noisy density has consistently led to solved structures where other methods fail. In this paper, an overview of the use of Rosetta for these difficult molecular-replacement problems is provided and new modeling developments that further improve model quality are described. Several variations to the method are introduced that significantly reduce the time needed to generate a model and the sampling required to improve the starting template. The improvements are benchmarked on a set of nine difficult cases and it is shown that this improved method obtains consistently better models in less running time. Finally, strategies for best using Rosetta to solve difficult molecular-replacement problems are presented and future directions for the role of structure-prediction methods in crystallography are discussed.
PMCID: PMC3817693  PMID: 24189231
structure prediction; molecular replacement; model building
25.  “Conditional Restraints”: Restraining the Free Atoms in ARP/wARP 
Structure(London, England:1993)  2009;17(2-3):183-189.
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.
PMCID: PMC2670983  PMID: 19217389

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