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1.  Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries 
The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described.
Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.
PMCID: PMC4051508  PMID: 24914984
protein–DNA complexes and macromolecule structure solutions; structure-solution pipelines; molecular replacement; density modification
2.  Extending molecular-replacement solutions with SHELXE  
Under favourable circumstances, density modification and polyalanine tracing with SHELXE can be used to improve and validate potential solutions from molecular replacement.
Although the program SHELXE was originally intended for the experimental phasing of macromolecules, it can also prove useful for expanding a small protein fragment to an almost complete polyalanine trace of the structure, given a favourable combination of native data resolution (better than about 2.1 Å) and solvent content. A correlation coefficient (CC) of more than 25% between the native structure factors and those calculated from the polyalanine trace appears to be a reliable indicator of success and has already been exploited in a number of pipelines. Here, a more detailed account of this usage of SHELXE for molecular-replacement solutions is given.
PMCID: PMC3817699  PMID: 24189237
molecular replacement; density modification; autotracing; SHELX
3.  Experimental phasing with SHELXC/D/E: combining chain tracing with density modification 
Experimental phasing with SHELXC/D/E has been enhanced by the incorporation of main-chain tracing into the iterative density modification; this also provides a simple and effective way of exploiting noncrystallographic symmetry.
The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue α-­helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a ‘no-go’ map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.
PMCID: PMC2852312  PMID: 20383001
experimental phasing of macromolecules; density modification; main-chain tracing; noncrystallographic symmetry; SHELX
4.  The restriction enzyme SgrAI: structure solution via combination of poor MIRAS and MR phases 
Phase information from both MIRAS and MR was used to produce an interpretable electron-density map of the novel type II restriction endonuclease SgrAI bound to DNA. The MR solution corrected an instructive error in the initially chosen averaging transformation.
Uninterpretable electron-density maps were obtained using either MIRAS phases or MR phases in attempts to determine the structure of the type II restriction endonuclease SgrAI bound to DNA. While neither solution strategy was particularly promising (map correlation coefficients of 0.29 and 0.22 with the final model, respectively, for the MIRAS and MR phases and Phaser Z scores of 4.0 and 4.3 for the rotation and translation searches), phase combination followed by density modification gave a readily interpretable map. MR with a distantly related model located a dimer in the asymmetric unit and provided the correct transformation to use in averaging electron density between SgrAI subunits. MIRAS data sets with low substitution and MR solutions from only distantly related models should not be ignored, as poor-quality starting phases can be significantly improved. The bootstrapping strategy employed to improve the initial MIRAS phases is described.
PMCID: PMC2659886  PMID: 19307723
SgrAI; MIRAS; phase combination; molecular replacement; density averaging; restriction enzymes
5.  Solving structures of protein complexes by molecular replacement with Phaser  
Four case studies in using maximum-likelihood molecular replacement, as implemented in the program Phaser, to solve structures of protein complexes are described.
Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ‘tree search with pruning’ approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ‘automated MR’ mode and two case studies are used to show how to modify the automated search strategy for problematic cases.
PMCID: PMC2483468  PMID: 17164524
macromolecular crystallography; molecular replacement; maximum likelihood
6.  Experimental phasing: best practice and pitfalls 
The pitfalls of experimental phasing are described.
Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces com­bined real–imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the software Phaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods.
PMCID: PMC2852310  PMID: 20382999
enantiomers; handedness; absolute configuration; chirality; twinning; experimental phasing
7.  Phaser crystallographic software 
Journal of Applied Crystallography  2007;40(Pt 4):658-674.
A description is given of Phaser-2.1: software for phasing macromolecular crystal structures by molecular replacement and single-wavelength anomalous dispersion phasing.
Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods. The novel phasing algorithms implemented in Phaser have been developed using maximum likelihood and multivariate statistics. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise, and for single-wavelength anomalous dispersion experimental phasing, the new algorithms, which account for correlations between F + and F −, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallographic community.
PMCID: PMC2483472  PMID: 19461840
computer programs; molecular replacement; SAD phasing; likelihood; structural genomics
8.  Using SAD data in Phaser  
SAD data can be used in Phaser to solve novel structures, supplement molecular-replacement phase information or identify anomalous scatterers from a final refined model.
Phaser is a program that implements likelihood-based methods to solve macromolecular crystal structures, currently by molecular replacement or single-wavelength anomalous diffraction (SAD). SAD phasing is based on a likelihood target derived from the joint probability distribution of observed and calculated pairs of Friedel-related structure factors. This target combines information from the total structure factor (primarily non-anomalous scattering) and the difference between the Friedel mates (anomalous scattering). Phasing starts from a substructure, which is usually but not necessarily a set of anomalous scatterers. The substructure can also be a protein model, such as one obtained by molecular replacement. Additional atoms are found using a log-likelihood gradient map, which shows the sites where the addition of scattering from a particular atom type would improve the likelihood score. An automated completion algorithm adds new sites, choosing optionally among different atom types, adds anisotropic B-factor parameters if appropriate and deletes atoms that refine to low occupancy. Log-likelihood gradient maps can also identify which atoms in a refined protein structure are anomalous scatterers, such as metal or halide ions. These maps are more sensitive than conventional model-phased anomalous difference Fouriers and the iterative completion algorithm is able to find a significantly larger number of convincing sites.
PMCID: PMC3069749  PMID: 21460452
SAD phasing; likelihood; molecular replacement
9.  Automated identification of elemental ions in macromolecular crystal structures 
The solvent-picking procedure in phenix.refine has been extended and combined with Phaser anomalous substructure completion and analysis of coordination geometry to identify and place elemental ions.
Many macromolecular model-building and refinement programs can automatically place solvent atoms in electron density at moderate-to-high resolution. This process frequently builds water molecules in place of elemental ions, the identification of which must be performed manually. The solvent-picking algorithms in phenix.refine have been extended to build common ions based on an analysis of the chemical environment as well as physical properties such as occupancy, B factor and anomalous scattering. The method is most effective for heavier elements such as calcium and zinc, for which a majority of sites can be placed with few false positives in a diverse test set of structures. At atomic resolution, it is observed that it can also be possible to identify tightly bound sodium and magnesium ions. A number of challenges that contribute to the difficulty of completely automating the process of structure completion are discussed.
PMCID: PMC3975891  PMID: 24699654
refinement; ions; PHENIX
10.  From electron microscopy to X-ray crystallography: molecular-replacement case studies 
Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement using various standard MR packages such as AMoRe, MOLREP and Phaser.
Multi-component molecular complexes are increasingly being tackled by structural biology, bringing X-ray crystallography into the purview of electron-microscopy (EM) studies. X-ray crystallography can utilize a low-resolution EM map for structure determination followed by phase extension to high resolution. Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement (MR) using various standard MR packages such as AMoRe, MOLREP and Phaser. The results demonstrate that EM maps are viable models for molecular replacement. Possible difficulties in data analysis, such as the effects of the EM magnification error, and the effect of MR positional/rotational errors on phase extension are discussed.
PMCID: PMC2394795  PMID: 18094470
electron microscopy; molecular replacement
11.  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
12.  Phaser.MRage: automated molecular replacement 
The functionality of the molecular-replacement pipeline phaser.MRage is introduced and illustrated with examples.
Phaser.MRage is a molecular-replacement automation framework that implements a full model-generation workflow and provides several layers of model exploration to the user. It is designed to handle a large number of models and can distribute calculations efficiently onto parallel hardware. In addition, phaser.MRage can identify correct solutions and use this information to accelerate the search. Firstly, it can quickly score all alternative models of a component once a correct solution has been found. Secondly, it can perform extensive analysis of identified solutions to find protein assemblies and can employ assembled models for subsequent searches. Thirdly, it is able to use a priori assembly information (derived from, for example, homologues) to speculatively place and score molecules, thereby customizing the search procedure to a certain class of protein molecule (for example, antibodies) and incorporating additional biological information into molecular replacement.
PMCID: PMC3817702  PMID: 24189240
molecular replacement; pipeline; automation; phaser.MRage
13.  Aqueous Solvation of Polyalanine α-Helices with Specific Water Molecules and with the CPCM and SM5.2 Aqueous Continuum Models using Density Functional Theory 
The Journal of Physical Chemistry. B  2012;116(4):1437-1445.
We present density functional theory (DFT) calculations at the X3LYP/D95(d,p) level on the solvation of polyalanine α-helices in water. The study includes the effects of discrete water molecules and the CPCM and AMSOL SM5.2 solvent continuum model both separately and in combination. We find that individual water molecules cooperatively hydrogen-bond to both the C- and N-termini of the helix, which results in increases in the dipole moment of the helix/water complex to more than the vector sum of their individual dipole moments. These waters are found to be more stable than in bulk solvent. On the other hand, individual water that interact with the backbone lower the dipole moment of the helix/water complex to below that of the helix, itself. Small clusters of waters at the termini increase the dipole moments of the helix/water aggregates, but the effect diminishes as more waters are added. We discuss the somewhat complex behavior of the helix with the discrete waters in the continuum models.
PMCID: PMC3271136  PMID: 22201227
14.  Fitting molecular fragments into electron density 
A number of techniques for the location of small and medium-sized model fragments in experimentally phased electron-density maps are explored. The application of one of these techniques to automated model building is discussed.
Molecular replacement is a powerful tool for the location of large models using structure-factor magnitudes alone. When phase information is available, it becomes possible to locate smaller fragments of the structure ranging in size from a few atoms to a single domain. The calculation is demanding, requiring a six-dimensional rotation and translation search. A number of approaches have been developed to this problem and a selection of these are reviewed in this paper. The application of one of these techniques to the problem of automated model building is explored in more detail, with particular reference to the problem of sequencing a protein main-chain trace.
PMCID: PMC2394793  PMID: 18094471
model fragments; electron-density maps; model building
15.  The Effects of Regularly Spaced Glutamine Substitutions on Alpha-Helical Peptide Structures. A DFT/ONIOM Study 
Chemical physics letters  2011;512(4-6):255-257.
The side-chains of the residues of glutamine (Q) and asparagine (N) contain amide groups. These can H-bond to each other in patterns similar to those of the backbone amides in α-helices. We show that mutating multiple Q's for alanines (A's) in a polyalanine helix stabilizes the helical structure, while similar mutations with multiple N's do not. We suggest that modification of peptides by incorporating Q's in such positions can make more robust helices that can be used to test the effects of secondary structures in biochemical experiments linked to proteins with variable structures such as tau and α-synuclein.
PMCID: PMC3171806  PMID: 21927063
16.  Effects of lysine substitution on stability of polyalanine alpha-helix 
We have studied stability of polyalanine alpha-helices with lysine residues added at C-and N-termini in gas-phase and aqueous solution. Monte Carlo simulations with the fixed-charges OPLS-AA and our polarizable POSSIM force fields were carried out. The results of the simulations confirm previously observed phenomena of the helix being stable with the LYS residue on the C-terminus and losing its helical structure if the charged LYS residue is located at the N-terminus of the polypeptide in gas-hase. Both OPLS-AA and POSSIM force fields performed essentially similarly, thus validity of the both for reproducing and predicting structures of such polypeptides has been confirmed. We have also studied the effect of replacing the normal N- and C-termini with methyl capping (this approach is often used in computational studies). Our results have demonstrated that the structure and stability of the polypeptides do not depend significantly on such a substitution although details of the resulting structure may change. The liquid-state simulations produced stable alpha-helixes regardless of the position of the protonated lysine residue. Overall, we have validated our polarizable POSSIM force field and the techniques used in the simulations, since the change of the helix structure as a function of the position of the LYS residue depends on a fine balance of energy contributions, and our methodology reproduced this balance well.
PMCID: PMC3591470  PMID: 23483820
force fields; electrostatic polarization; alpha-helix; gas-phase peptide conformation; polyalanine
17.  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
18.  Extending the PRIME Model for Protein Aggregation to All Twenty Amino Acids 
Proteins  2010;78(14):2950-2960.
We extend PRIME, an intermediate-resolution protein model previously used in simulations of the aggregation of polyalanine and polyglutamine, to the description of the geometry and energetics of peptides containing all twenty amino acid residues. The 20 amino acid side chains are classified into 14 groups according to their hydrophobicity, polarity, size, charge and potential for side chain hydrogen bonding. The parameters for extended PRIME, called PRIME 20, include hydrogen-bonding energies, side-chain interaction range and energy, and excluded volume. The parameters are obtained by applying a perceptron- learning algorithm and a modified stochastic learning algorithm that optimizes the energy gap between 711 known native states from the PDB and decoy structures generated by gapless threading. The number of independent pair-interaction parameters is chosen to be small enough to be physically meaningful yet large enough to give reasonably accurate results in discriminating decoys from native structures. The most physically meaningful results are obtained with 19 energy parameters.
PMCID: PMC2945877  PMID: 20740494
19.  V-Phaser 2: variant inference for viral populations 
BMC Genomics  2013;14:674.
Massively parallel sequencing offers the possibility of revolutionizing the study of viral populations by providing ultra deep sequencing (tens to hundreds of thousand fold coverage) of complete viral genomes. However, differentiation of true low frequency variants from sequencing errors remains challenging.
We developed a software package, V-Phaser 2, for inferring intrahost diversity within viral populations. This program adds three major new methodologies to the state of the art: a technique to efficiently utilize paired end read data for calling phased variants, a new strategy to represent and infer length polymorphisms, and an in line filter for erroneous calls arising from systematic sequencing artifacts. We have also heavily optimized memory and run time performance. This combination of algorithmic and technical advances allows V-Phaser 2 to fully utilize extremely deep paired end sequencing data (such as generated by Illumina sequencers) to accurately infer low frequency intrahost variants in viral populations in reasonable time on a standard desktop computer. V-Phaser 2 was validated and compared to both QuRe and the original V-Phaser on three datasets obtained from two viral populations: a mixture of eight known strains of West Nile Virus (WNV) sequenced on both 454 Titanium and Illumina MiSeq and a mixture of twenty-four known strains of WNV sequenced only on 454 Titanium. V-Phaser 2 outperformed the other two programs in both sensitivity and specificity while using more than five fold less time and memory.
We developed V-Phaser 2, a publicly available software tool (V-Phaser 2 can be accessed via: and is freely available for academic use) that enables the efficient analysis of ultra-deep sequencing data produced by common next generation sequencing platforms for viral populations.
PMCID: PMC3907024  PMID: 24088188
Viral population; Variant calling; Length polymorphisms; Phasing; Next generation sequencing
20.  MH2c: Characterization of major histocompatibility α-helices – an information criterion approach☆ 
Computer Physics Communications  2012;183(7):1481-1490.
Major histocompatibility proteins share a common overall structure or peptide binding groove. Two binding groove domains, on the same chain for major histocompatibility class I or on two different chains for major histocompatibility class II, contribute to that structure that consists of two α-helices (“wall”) and a sheet of eight anti-parallel beta strands (“floor”). Apart from the peptide presented in the groove, the major histocompatibility α-helices play a central role for the interaction with the T cell receptor. This study presents a generalized mathematical approach for the characterization of these helices. We employed polynomials of degree 1 to 7 and splines with 1 to 2 nodes based on polynomials of degree 1 to 7 on the α-helices projected on their principal components. We evaluated all models with a corrected Akaike Information Criterion to determine which model represents the α-helices in the best way without overfitting the data. This method is applicable for both the stationary and the dynamic characterization of α-helices. By deriving differential geometric parameters from these models one obtains a reliable method to characterize and compare α-helices for a broad range of applications.
Program summary
Program title: MH2c (MH helix curves)
Catalogue identifier: AELX_v1_0
Program summary URL:
Program obtainable from: CPC Program Library, Queenʼs University, Belfast, N. Ireland
Licensing provisions: Standard CPC licence,
No. of lines in distributed program, including test data, etc.: 327 565
No. of bytes in distributed program, including test data, etc.: 17 433 656
Distribution format: tar.gz
Programming language: Matlab
Computer: Personal computer architectures
Operating system: Windows, Linux, Mac (all systems on which Matlab can be installed)
RAM: Depends on the trajectory size, min. 1 GB (Matlab)
Classification: 2.1, 4.9, 4.14
External routines: Curve Fitting Toolbox and Statistic Toolbox of Matlab
Nature of problem: Major histocompatibility (MH) proteins share a similar overall structure. However, identical MH alleles which present different peptides differ by subtle conformational alterations. One hypothesis is that such conformational differences could be another level of T cell regulation. By this software package we present a reliable and systematic way to compare different MH structures to each other.
Solution method: We tested several fitting approaches on all available experimental crystal structures of MH to obtain an overall picture of how to describe MH helices. For this purpose we transformed all complexes into the same space and applied splines and polynomials of several degrees to them. To draw a general conclusion which method fits them best we employed the “corrected Akaike Information Criterion”. The software is applicable for all kinds of helices of biomolecules.
Running time: Depends on the data, for a single stationary structure the runtime should not exceed a few seconds.
PMCID: PMC3617674  PMID: 23564964
MH2c, MH helix curves (name of software); TR, T cell receptor; p, peptide; MH, major histocompatibility; MH1, major histocompatibility class I; MH2, major histocompatibility class II; G, binding groove; CDR, complementarity determining region; MD, Molecular Dynamics; PDB, Protein Data Bank; VMD, Visual Molecular Dynamics; PCA, Principal Component Analysis; PC, principal component; AIC, Akaike Information Criterion; cAIC, corrected Akaike Information Criterion; IMGT®, the international ImMunoGeneTics information system®; MH; MHC; Helix; Akaike Information Criterion; Minimization and fitting; Utility; Structure and properties; Molecular dynamics simulation; Proteins; Secondary structure; Theory, modeling, and computer simulation; Conformational changes
21.  Refinement of macromolecular structures against neutron data with SHELXL2013  
Journal of Applied Crystallography  2013;47(Pt 1):462-466.
SHELXL2013 contains improvements over the previous versions that facilitate the refinement of macromolecular structures against neutron data. This article highlights several features of particular interest for this purpose and includes a list of restraints for H-atom refinement.
Some of the improvements in SHELX2013 make SHELXL convenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to use SHELXL for refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [Acta Cryst. (1991), A47, 392–400] restraints to H atoms and discusses several of the features of SHELXL that make the program particularly useful for the investigation of H atoms with neutron diffraction. SHELXL2013 is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures.
PMCID: PMC3937812  PMID: 24587788
single-crystal neutron diffraction; macromolecular structure refinement; hydrogen restraints; SHELXL2013
22.  SCEDS: protein fragments for molecular replacement in Phaser  
Protein fragments suitable for use in molecular replacement can be generated by normal-mode perturbation, analysis of the difference distance matrix of the original versus normal-mode perturbed structures, and SCEDS, a score that measures the sphericity, continuity, equality and density of the resulting fragments.
A method is described for generating protein fragments suitable for use as molecular-replacement (MR) template models. The template model for a protein suspected to undergo a conformational change is perturbed along combinations of low-frequency normal modes of the elastic network model. The unperturbed structure is then compared with each perturbed structure in turn and the structurally invariant regions are identified by analysing the difference distance matrix. These fragments are scored with SCEDS, which is a combined measure of the sphericity of the fragments, the continuity of the fragments with respect to the polypeptide chain, the equality in number of atoms in the fragments and the density of Cα atoms in the triaxial ellipsoid of the fragment extents. The fragment divisions with the highest SCEDS are then used as separate template models for MR. Test cases show that where the protein contains fragments that undergo a change in juxtaposition between template model and target, SCEDS can identify fragments that lead to a lower R factor after ten cycles of all-atom refinement with REFMAC5 than the original template structure. The method has been implemented in the software Phaser.
PMCID: PMC3817695  PMID: 24189233
difference distance matrix; normal-mode analysis
23.  Helix formation in the polymer brush 
Macromolecules  2011;44(12):4977-4987.
This work considers the physics of a brush formed by polymers capable of undergoing a helix-coil transition. A self-consistent field approximation for strongly stretched polymer chains is used in combination with a lattice model of the interaction energy in helix-coil mixtures. Crowding-induced chain stretching stabilizes helix formation at moderate tethering densities while high tethering density causes sufficiently strong stretching to unravel segments of the helix, resulting in distinct layers of monomer density and helical content. Compared to a random-coil brush at low-to-moderate tethering density, a helicogenic brush is less resistant to compression in the direction perpendicular to stretching due to easy alignment of helices and fewer unfavorable interactions between helical segments. At higher tethering density, the abovementioned stretch-induced decrease in helical content resists further compression. The proposed model is useful for understanding an emerging class of biomaterials that utilize helix-forming polymer brushes to induce shape changes or to stabilize biofunctional helical peptide sequences.
PMCID: PMC3140226  PMID: 21785512
24.  Crystallization and calcium/sulfur SAD phasing of the human EF-hand protein S100A2 
The structure of Ca2+-bound EF-hand protein S100A2 was determined by calcium and sulfur SAD at a wavelength of 0.90 Å.
Human S100A2 is an EF-hand protein and acts as a major tumour suppressor, binding and activating p53 in a Ca2+-dependent manner. Ca2+-bound S100A2 was crystallized and its structure was determined based on the anomalous scattering provided by six S atoms from methionine residues and four calcium ions present in the asymmetric unit. Although the diffraction data were recorded at a wavelength of 0.90 Å, which is usually not assumed to be suitable for calcium/sulfur SAD, the anomalous signal was satisfactory. A nine-atom substructure was determined at 1.8 Å resolution using SHELXD, and SHELXE was used for density modification and phase extension to 1.3 Å resolution. The electron-density map obtained was well interpretable and could be used for automated model building by ARP/wARP.
PMCID: PMC2935220  PMID: 20823519
S100A2; EF-hands; calcium; sulfur SAD
25.  Multilevel X-Pol: A Fragment-based Method with Mixed Quantum Mechanical Representations of Different Fragments 
The Journal of Physical Chemistry. B  2012;116(23):6781-6788.
The explicit polarization (X-Pol) method is a fragment-based quantum mechanical model, in which a macromolecular system in solution is partitioned into monomer fragments. The present study extends the original X-Pol method, where all fragments are treated using the same electronic structure theory, to a multilevel representations, called multilevel X-Pol, in which different electronic structure methods are used to describe different fragments. The multilevel X-Pol method has been implemented into Gaussian 09. A key ingredient that is used to couple interfragment electrostatic interactions at different levels of theory is the use of the response density for post-self-consistent-field energy (The response density is also called the generalized density). The method is useful for treating fragments in a small region of the system such as the solute molecules or the substrate and amino acids in the active site of an enzyme with a high-level theory, and the fragments in the rest of the system by a lower-level and computationally more efficient method. The method is illustrated here by applications to hydrogen bonding complexes in which one fragment is treated with the hybrid M06 density functional, Møller-Plesset perturbation theory, or coupled cluster theory, and the other fragments are treated by Hartree-Fock theory or the B3LYP or M06 hybrid density functionals.
PMCID: PMC3376169  PMID: 22428657
Mixed fragment method; explicit polarization theory; fragment-based molecular orbital; block-localized density functional theory

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