Publications by authors named "Robert A Nicholls"

21 Publications

  • Page 1 of 1

The missing link: covalent linkages in structural models.

Acta Crystallogr D Struct Biol 2021 Jun 19;77(Pt 6):727-745. Epub 2021 May 19.

Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

Covalent linkages between constituent blocks of macromolecules and ligands have been subject to inconsistent treatment during the model-building, refinement and deposition process. This may stem from a number of sources, including difficulties with initially detecting the covalent linkage, identifying the correct chemistry, obtaining an appropriate restraint dictionary and ensuring its correct application. The analysis presented herein assesses the extent of problems involving covalent linkages in the Protein Data Bank (PDB). Not only will this facilitate the remediation of existing models, but also, more importantly, it will inform and thus improve the quality of future linkages. By considering linkages of known type in the CCP4 Monomer Library (CCP4-ML), failure to model a covalent linkage is identified to result in inaccurate (systematically longer) interatomic distances. Scanning the PDB for proximal atom pairs that do not have a corresponding type in the CCP4-ML reveals a large number of commonly occurring types of unannotated potential linkages; in general, these may or may not be covalently linked. Manual consideration of the most commonly occurring cases identifies a number of genuine classes of covalent linkages. The recent expansion of the CCP4-ML is discussed, which has involved the addition of over 16 000 and the replacement of over 11 000 component dictionaries using AceDRG. As part of this effort, the CCP4-ML has also been extended using AceDRG link dictionaries for the aforementioned linkage types identified in this analysis. This will facilitate the identification of such linkage types in future modelling efforts, whilst concurrently easing the process involved in their application. The need for a universal standard for maintaining link records corresponding to covalent linkages, and references to the associated dictionaries used during modelling and refinement, following deposition to the PDB is emphasized. The importance of correctly modelling covalent linkages is demonstrated using a case study, which involves the covalent linkage of an inhibitor to the main protease in various viral species, including SARS-CoV-2. This example demonstrates the importance of properly modelling covalent linkages using a comprehensive restraint dictionary, as opposed to just using a single interatomic distance restraint or failing to model the covalent linkage at all.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798321003934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8171067PMC
June 2021

Modelling covalent linkages in CCP4.

Acta Crystallogr D Struct Biol 2021 Jun 19;77(Pt 6):712-726. Epub 2021 May 19.

Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

In this contribution, the current protocols for modelling covalent linkages within the CCP4 suite are considered. The mechanism used for modelling covalent linkages is reviewed: the use of dictionaries for describing changes to stereochemistry as a result of the covalent linkage and the application of link-annotation records to structural models to ensure the correct treatment of individual instances of covalent linkages. Previously, linkage descriptions were lacking in quality compared with those of contemporary component dictionaries. Consequently, AceDRG has been adapted for the generation of link dictionaries of the same quality as for individual components. The approach adopted by AceDRG for the generation of link dictionaries is outlined, which includes associated modifications to the linked components. A number of tools to facilitate the practical modelling of covalent linkages available within the CCP4 suite are described, including a new restraint-dictionary accumulator, the Make Covalent Link tool and AceDRG interface in Coot, the 3D graphical editor JLigand and the mechanisms for dealing with covalent linkages in the CCP4i2 and CCP4 Cloud environments. These integrated solutions streamline and ease the covalent-linkage modelling workflow, seamlessly transferring relevant information between programs. Current recommended practice is elucidated by means of instructive practical examples. By summarizing the different approaches to modelling linkages that are available within the CCP4 suite, limitations and potential pitfalls that may be encountered are highlighted in order to raise awareness, with the intention of improving the quality of future modelled covalent linkages in macromolecular complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798321001753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8171069PMC
June 2021

Current approaches for the fitting and refinement of atomic models into cryo-EM maps using CCP-EM.

Acta Crystallogr D Struct Biol 2018 Jun 30;74(Pt 6):492-505. Epub 2018 May 30.

Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England.

Recent advances in instrumentation and software have resulted in cryo-EM rapidly becoming the method of choice for structural biologists, especially for those studying the three-dimensional structures of very large macromolecular complexes. In this contribution, the tools available for macromolecular structure refinement into cryo-EM reconstructions that are available via CCP-EM are reviewed, specifically focusing on REFMAC5 and related tools. Whilst originally designed with a view to refinement against X-ray diffraction data, some of these tools have been able to be repurposed for cryo-EM owing to the same principles being applicable to refinement against cryo-EM maps. Since both techniques are used to elucidate macromolecular structures, tools encapsulating prior knowledge about macromolecules can easily be transferred. However, there are some significant qualitative differences that must be acknowledged and accounted for; relevant differences between these techniques are highlighted. The importance of phases is considered and the potential utility of replacing inaccurate amplitudes with their expectations is justified. More pragmatically, an upper bound on the correlation between observed and calculated Fourier coefficients, expressed in terms of the Fourier shell correlation between half-maps, is demonstrated. The importance of selecting appropriate levels of map blurring/sharpening is emphasized, which may be facilitated by considering the behaviour of the average map amplitude at different resolutions, as well as the utility of simultaneously viewing multiple blurred/sharpened maps. Features that are important for the purposes of computational efficiency are discussed, notably the Divide and Conquer pipeline for the parallel refinement of large macromolecular complexes. Techniques that have recently been developed or improved in Coot to facilitate and expedite the building, fitting and refinement of atomic models into cryo-EM maps are summarized. Finally, a tool for symmetry identification from a given map or coordinate set, ProSHADE, which can identify the point group of a map and thus may be used during deposition as well as during molecular visualization, is introduced.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798318007313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096485PMC
June 2018

CCP4i2: the new graphical user interface to the CCP4 program suite.

Acta Crystallogr D Struct Biol 2018 02 1;74(Pt 2):68-84. Epub 2018 Feb 1.

STFC Rutherford Appleton Laboratory, Chilton, Didcot OX11 0QX, England.

The CCP4 (Collaborative Computational Project, Number 4) software suite for macromolecular structure determination by X-ray crystallography groups brings together many programs and libraries that, by means of well established conventions, interoperate effectively without adhering to strict design guidelines. Because of this inherent flexibility, users are often presented with diverse, even divergent, choices for solving every type of problem. Recently, CCP4 introduced CCP4i2, a modern graphical interface designed to help structural biologists to navigate the process of structure determination, with an emphasis on pipelining and the streamlined presentation of results. In addition, CCP4i2 provides a framework for writing structure-solution scripts that can be built up incrementally to create increasingly automatic procedures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798317016035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947771PMC
February 2018

Overview of refinement procedures within REFMAC5: utilizing data from different sources.

Acta Crystallogr D Struct Biol 2018 03 2;74(Pt 3):215-227. Epub 2018 Mar 2.

Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, England.

Refinement is a process that involves bringing into agreement the structural model, available prior knowledge and experimental data. To achieve this, the refinement procedure optimizes a posterior conditional probability distribution of model parameters, including atomic coordinates, atomic displacement parameters (B factors), scale factors, parameters of the solvent model and twin fractions in the case of twinned crystals, given observed data such as observed amplitudes or intensities of structure factors. A library of chemical restraints is typically used to ensure consistency between the model and the prior knowledge of stereochemistry. If the observation-to-parameter ratio is small, for example when diffraction data only extend to low resolution, the Bayesian framework implemented in REFMAC5 uses external restraints to inject additional information extracted from structures of homologous proteins, prior knowledge about secondary-structure formation and even data obtained using different experimental methods, for example NMR. The refinement procedure also generates the `best' weighted electron-density maps, which are useful for further model (re)building. Here, the refinement of macromolecular structures using REFMAC5 and related tools distributed as part of the CCP4 suite is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798318000979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947762PMC
March 2018

AUSPEX: a graphical tool for X-ray diffraction data analysis.

Acta Crystallogr D Struct Biol 2017 Sep 8;73(Pt 9):729-737. Epub 2017 Aug 8.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, England.

In this paper, AUSPEX, a new software tool for experimental X-ray data analysis, is presented. Exploring the behaviour of diffraction intensities and the associated estimated uncertainties facilitates the discovery of underlying problems and can help users to improve their data acquisition and processing in order to obtain better structural models. The program enables users to inspect the distribution of observed intensities (or amplitudes) against resolution as well as the associated estimated uncertainties (sigmas). It is demonstrated how AUSPEX can be used to visually and automatically detect ice-ring artefacts in integrated X-ray diffraction data. Such artefacts can hamper structure determination, but may be difficult to identify from the raw diffraction images produced by modern pixel detectors. The analysis suggests that a significant portion of the data sets deposited in the PDB contain ice-ring artefacts. Furthermore, it is demonstrated how other problems in experimental X-ray data caused, for example, by scaling and data-conversion procedures can be detected by AUSPEX.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S205979831700969XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586246PMC
September 2017

Low Resolution Refinement of Atomic Models Against Crystallographic Data.

Methods Mol Biol 2017 ;1607:565-593

MRC Laboratory of Molecular Biology, Francis Crick Avenue, CB2 0QH, Cambridge, UK.

This review describes some of the problems encountered during low-resolution refinement and map calculation. Refinement is considered as an application of Bayes' theorem, allowing combination of information from various sources including crystallographic experimental data and prior chemical and structural knowledge. The sources of prior knowledge relevant to macromolecules include basic chemical information such as bonds and angles, structural information from reference models of known homologs, knowledge about secondary structures, hydrogen bonding patterns, and similarity of non-crystallographically related copies of a molecule. Additionally, prior information encapsulating local conformational conservation is exploited, keeping local interatomic distances similar to those in the starting atomic model. The importance of designing an accurate likelihood function-the only link between model parameters and observed data-is emphasized. The review also reemphasizes the importance of phases, and describes how the use of raw observed amplitudes could give a better correlation between the calculated and "true" maps. It is shown that very noisy or absent observations can be replaced by calculated structure factors, weighted according to the accuracy of the atomic model. This approach helps to smoothen the map. However, such replacement should be used sparingly, as the bias toward errors in the model could be too much to avoid. It is in general recommended that, whenever a new map is calculated, map quality should be judged by inspection of the parts of the map where there is no atomic model. It is also noted that it is advisable to work with multiple blurred and sharpened maps, as different parts of a crystal may exhibit different degrees of mobility. Doing so can allow accurate building of atomic models, accounting for overall shape as well as finer structural details. Some of the results described in this review have been implemented in the programs REFMAC5, ProSMART and LORESTR, which are available as part of the CCP4 software suite.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7000-1_23DOI Listing
March 2018

Ligand fitting with CCP4.

Acta Crystallogr D Struct Biol 2017 02 1;73(Pt 2):158-170. Epub 2017 Feb 1.

Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England.

Crystal structures of protein-ligand complexes are often used to infer biology and inform structure-based drug discovery. Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective protein-ligand complex. This paper discusses key stages in the ligand-fitting process, including ligand binding-site identification, ligand description and conformer generation, ligand fitting, refinement and subsequent validation. The CCP4 suite contains a number of software tools that facilitate this task: AceDRG for the creation of ligand descriptions and conformers, Lidia and JLigand for two-dimensional and three-dimensional ligand editing and visual analysis, Coot for density interpretation, ligand fitting, analysis and validation, and REFMAC5 for macromolecular refinement. In addition to recent advancements in automatic carbohydrate building in Coot (LO/Carb) and ligand-validation tools (FLEV), the release of the CCP4i2 GUI provides an integrated solution that streamlines the ligand-fitting workflow, seamlessly passing results from one program to the next. The ligand-fitting process is illustrated using instructive practical examples, including problematic cases such as post-translational modifications, highlighting the need for careful analysis and rigorous validation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798316020143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297919PMC
February 2017

AceDRG: a stereochemical description generator for ligands.

Acta Crystallogr D Struct Biol 2017 02 1;73(Pt 2):112-122. Epub 2017 Feb 1.

Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England.

The program AceDRG is designed for the derivation of stereochemical information about small molecules. It uses local chemical and topological environment-based atom typing to derive and organize bond lengths and angles from a small-molecule database: the Crystallography Open Database (COD). Information about the hybridization states of atoms, whether they belong to small rings (up to seven-membered rings), ring aromaticity and nearest-neighbour information is encoded in the atom types. All atoms from the COD have been classified according to the generated atom types. All bonds and angles have also been classified according to the atom types and, in a certain sense, bond types. Derived data are tabulated in a machine-readable form that is freely available from CCP4. AceDRG can also generate stereochemical information, provided that the basic bonding pattern of a ligand is known. The basic bonding pattern is perceived from one of the computational chemistry file formats, including SMILES, mmCIF, SDF MOL and SYBYL MOL2 files. Using the bonding chemistry, atom types, and bond and angle tables generated from the COD, AceDRG derives the `ideal' bond lengths, angles, plane groups, aromatic rings and chirality information, and writes them to an mmCIF file that can be used by the refinement program REFMAC5 and the model-building program Coot. Other refinement and model-building programs such as PHENIX and BUSTER can also use these files. AceDRG also generates one or more coordinate sets corresponding to the most favourable conformation(s) of a given ligand. AceDRG employs RDKit for chemistry perception and for initial conformation generation, as well as for the interpretation of SMILES strings, SDF MOL and SYBYL MOL2 files.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798317000067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297914PMC
February 2017

Validation and extraction of molecular-geometry information from small-molecule databases.

Acta Crystallogr D Struct Biol 2017 02 1;73(Pt 2):103-111. Epub 2017 Feb 1.

Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England.

A freely available small-molecule structure database, the Crystallography Open Database (COD), is used for the extraction of molecular-geometry information on small-molecule compounds. The results are used for the generation of new ligand descriptions, which are subsequently used by macromolecular model-building and structure-refinement software. To increase the reliability of the derived data, and therefore the new ligand descriptions, the entries from this database were subjected to very strict validation. The selection criteria made sure that the crystal structures used to derive atom types, bond and angle classes are of sufficiently high quality. Any suspicious entries at a crystal or molecular level were removed from further consideration. The selection criteria included (i) the resolution of the data used for refinement (entries solved at 0.84 Å resolution or higher) and (ii) the structure-solution method (structures must be from a single-crystal experiment and all atoms of generated molecules must have full occupancies), as well as basic sanity checks such as (iii) consistency between the valences and the number of connections between atoms, (iv) acceptable bond-length deviations from the expected values and (v) detection of atomic collisions. The derived atom types and bond classes were then validated using high-order moment-based statistical techniques. The results of the statistical analyses were fed back to fine-tune the atom typing. The developed procedure was repeated four times, resulting in fine-grained atom typing, bond and angle classes. The procedure will be repeated in the future as and when new entries are deposited in the COD. The whole procedure can also be applied to any source of small-molecule structures, including the Cambridge Structural Database and the ZINC database.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798317000079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297913PMC
February 2017

Automated refinement of macromolecular structures at low resolution using prior information.

Acta Crystallogr D Struct Biol 2016 10 30;72(Pt 10):1149-1161. Epub 2016 Sep 30.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, England.

Since the ratio of the number of observations to adjustable parameters is small at low resolution, it is necessary to use complementary information for the analysis of such data. ProSMART is a program that can generate restraints for macromolecules using homologous structures, as well as generic restraints for the stabilization of secondary structures. These restraints are used by REFMAC5 to stabilize the refinement of an atomic model. However, the optimal refinement protocol varies from case to case, and it is not always obvious how to select appropriate homologous structure(s), or other sources of prior information, for restraint generation. After running extensive tests on a large data set of low-resolution models, the best-performing refinement protocols and strategies for the selection of homologous structures have been identified. These strategies and protocols have been implemented in the Low-Resolution Structure Refinement (LORESTR) pipeline. The pipeline performs auto-detection of twinning and selects the optimal scaling method and solvent parameters. LORESTR can either use user-supplied homologous structures, or run an automated BLAST search and download homologues from the PDB. The pipeline executes multiple model-refinement instances using different parameters in order to find the best protocol. Tests show that the automated pipeline improves R factors, geometry and Ramachandran statistics for 94% of the low-resolution cases from the PDB included in the test set.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798316014534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053141PMC
October 2016

Diagnostic accuracy for major depression in multiple sclerosis using self-report questionnaires.

Brain Behav 2015 Sep 14;5(9):e00365. Epub 2015 Jul 14.

Center for Molecular Neurobiology, Institute of Neuroimmunology and Multiple Sclerosis (INIMS), University Medical Center Hamburg-Eppendorf Hamburg, Germany ; Department of Medical Psychology, University Medical Center Hamburg-Eppendorf Hamburg, Germany ; Department of Psychiatry and Psychotherapy, Campus Benjamin Franklin (CBF), Charité Universitätsmedizin Berlin, Germany.

Objective: Multiple sclerosis and major depressive disorder frequently co-occur but depression often remains undiagnosed in this population. Self-rated depression questionnaires are a good option where clinician-based standardized diagnostics are not feasible. However, there is a paucity of data on diagnostic accuracy of self-report measures for depression in multiple sclerosis (MS). Moreover, head-to-head comparisons of common questionnaires are largely lacking. This could be particularly relevant for high-risk patients with depressive symptoms. Here, we compare the diagnostic accuracy of the Beck Depression Inventory (BDI) and 30-item version of the Inventory of Depressive Symptomatology Self-Rated (IDS-SR30) for major depressive disorder (MSS) against diagnosis by a structured clinical interview.

Methods: Patients reporting depressive symptoms completed the BDI, the IDS-SR30 and underwent diagnostic assessment (Mini International Neuropsychiatric Interview, M.I.N.I.). Receiver-Operating Characteristic analyses were performed, providing error estimates and false-positive/negative rates of suggested thresholds.

Results: Data from n = 31 MS patients were available. BDI and IDS-SR30 total score were significantly correlated (r = 0.82). The IDS-SR30total score, cognitive subscore, and BDI showed excellent to good accuracy (area under the curve (AUC) 0.86, 0.91, and 0.85, respectively).

Conclusion: Both the IDS-SR30 and the BDI are useful to quantify depressive symptoms showing good sensitivity and specificity. The IDS-SR30 cognitive subscale may be useful as a screening tool and to quantify affective/cognitive depressive symptomatology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/brb3.365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589811PMC
September 2015

MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA.

Elife 2015 Jul 11;4:e06744. Epub 2015 Jul 11.

Division of Biochemistry and CGC.nl, Netherlands Cancer Institute, Amsterdam, Netherlands.

To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.06744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521584PMC
July 2015

Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions.

Acta Crystallogr D Biol Crystallogr 2015 Jan 1;71(Pt 1):136-53. Epub 2015 Jan 1.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England.

The recent rapid development of single-particle electron cryo-microscopy (cryo-EM) now allows structures to be solved by this method at resolutions close to 3 Å. Here, a number of tools to facilitate the interpretation of EM reconstructions with stereochemically reasonable all-atom models are described. The BALBES database has been repurposed as a tool for identifying protein folds from density maps. Modifications to Coot, including new Jiggle Fit and morphing tools and improved handling of nucleic acids, enhance its functionality for interpreting EM maps. REFMAC has been modified for optimal fitting of atomic models into EM maps. As external structural information can enhance the reliability of the derived atomic models, stabilize refinement and reduce overfitting, ProSMART has been extended to generate interatomic distance restraints from nucleic acid reference structures, and a new tool, LIBG, has been developed to generate nucleic acid base-pair and parallel-plane restraints. Furthermore, restraint generation has been integrated with visualization and editing in Coot, and these restraints have been applied to both real-space refinement in Coot and reciprocal-space refinement in REFMAC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004714021683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304694PMC
January 2015

Data to knowledge: how to get meaning from your result.

IUCrJ 2015 Jan 1;2(Pt 1):45-58. Epub 2015 Jan 1.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.

Structural and functional studies require the development of sophisticated 'Big Data' technologies and software to increase the knowledge derived and ensure reproducibility of the data. This paper presents summaries of the Structural Biology Knowledge Base, the VIPERdb Virus Structure Database, evaluation of homology modeling by the Protein Model Portal, the ProSMART tool for conformation-independent structure comparison, the LabDB 'super' laboratory information management system and the Cambridge Structural Database. These techniques and technologies represent important tools for the transformation of crystallographic data into knowledge and information, in an effort to address the problem of non-reproducibility of experimental results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2052252514023306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285880PMC
January 2015

Conformation-independent structural comparison of macromolecules with ProSMART.

Acta Crystallogr D Biol Crystallogr 2014 Sep 29;70(Pt 9):2487-99. Epub 2014 Aug 29.

Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, England.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004714016241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157452PMC
September 2014

SCEDS: protein fragments for molecular replacement in Phaser.

Acta Crystallogr D Biol Crystallogr 2013 Nov 4;69(Pt 11):2216-25. Epub 2013 Oct 4.

Cambridge Institute for Medical Research, Department of Haematology, University of Cambridge, Hills Road, Cambridge CB2 0XY, England.

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 C(α) 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0907444913021811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817695PMC
November 2013

Low-resolution refinement tools in REFMAC5.

Acta Crystallogr D Biol Crystallogr 2012 Apr 16;68(Pt 4):404-17. Epub 2012 Mar 16.

Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, England.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S090744491105606XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322599PMC
April 2012

Decreased hydrocortisone sensitivity of T cell function in multiple sclerosis-associated major depression.

Psychoneuroendocrinology 2012 Oct 27;37(10):1712-8. Epub 2012 Mar 27.

Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center for Molecular Neurobiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS with a high prevalence of depression. Both MS and depression have been linked to elevated cortisol levels and inflammation, indicating disturbed endocrine-immune regulation. An imbalance in mineralocorticoid versus glucocorticoid signaling in the CNS has been proposed as a pathogenetic mechanism of depression. Intriguingly, both receptors are also expressed in lymphocytes, but their role for 'escape' of the immune system from endocrine control is unknown. Using steroid sensitivity of T cell function as a read-out system, we here investigate a potential role of mineralocorticoid receptor (MR) versus glucocorticoid receptor (GR) regulation in the immune system as a biological mechanism underlying MS-associated major depression. Twelve female MS patients meeting diagnostic criteria for current major depressive disorder (MDD) were compared to twelve carefully matched MS patients without depression. We performed lymphocyte phenotyping by flow cytometry. In addition, steroid sensitivity of T cell proliferation was tested using hydrocortisone as well as MR (aldosterone) and GR (dexamethasone) agonists. Sensitivity to hydrocortisone was decreased in T cells from depressed MS patients. Experiments with agonists suggested disturbed MR regulation, but intact GR function. Importantly, there were no differences in lymphocyte composition and frequency of T cell subsets, indicating that the differences in steroid sensitivity are unlikely to be secondary to shifts in the immune compartment. To our knowledge, this study provides first evidence for altered steroid sensitivity of T cells from MS patients with comorbid MDD possibly due to MR dysregulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.psyneuen.2012.03.001DOI Listing
October 2012

Structural characterization of Helicobacter pylori dethiobiotin synthetase reveals differences between family members.

FEBS J 2012 Mar 27;279(6):1093-105. Epub 2012 Feb 27.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.

Dethiobiotin synthetase (DTBS) is involved in the biosynthesis of biotin in bacteria, fungi, and plants. As humans lack this pathway, DTBS is a promising antimicrobial drug target. We determined structures of DTBS from Helicobacter pylori (hpDTBS) bound with cofactors and a substrate analog, and described its unique characteristics relative to other DTBS proteins. Comparison with bacterial DTBS orthologs revealed considerable structural differences in nucleotide recognition. The C-terminal region of DTBS proteins, which contains two nucleotide-recognition motifs, differs greatly among DTBS proteins from different species. The structure of hpDTBS revealed that this protein is unique and does not contain a C-terminal region containing one of the motifs. The single nucleotide-binding motif in hpDTBS is similar to its counterpart in GTPases; however, isothermal titration calorimetry binding studies showed that hpDTBS has a strong preference for ATP. The structural determinants of ATP specificity were assessed with X-ray crystallographic studies of hpDTBS·ATP and hpDTBS·GTP complexes. The unique mode of nucleotide recognition in hpDTBS makes this protein a good target for H. pylori-specific inhibitors of the biotin synthesis pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2012.08506.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392494PMC
March 2012

REFMAC5 for the refinement of macromolecular crystal structures.

Acta Crystallogr D Biol Crystallogr 2011 Apr 18;67(Pt 4):355-67. Epub 2011 Mar 18.

Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, England.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0907444911001314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069751PMC
April 2011