Publications by authors named "Garib N Murshudov"

51 Publications

Local and global analysis of macromolecular atomic displacement parameters.

Acta Crystallogr D Struct Biol 2020 Oct 22;76(Pt 10):926-937. Epub 2020 Sep 22.

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

This paper describes the global and local analysis of atomic displacement parameters (ADPs) of macromolecules in X-ray crystallography. The distribution of ADPs is shown to follow the shifted inverse-gamma distribution or a mixture of these distributions. The mixture parameters are estimated using the expectation-maximization algorithm. In addition, a method for the resolution- and individual ADP-dependent local analysis of neighbouring atoms has been designed. This method facilitates the detection of mismodelled atoms, heavy-metal atoms and disordered and/or incorrectly modelled ligands. Both global and local analyses can be used to detect errors in atomic models, thus helping in the (re)building, refinement and validation of macromolecular structures. This method can also serve as an additional validation tool during PDB deposition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798320011043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543658PMC
October 2020

The predictive power of data-processing statistics.

IUCrJ 2020 Mar 27;7(Pt 2):342-354. Epub 2020 Feb 27.

Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.

This study describes a method to estimate the likelihood of success in determining a macromolecular structure by X-ray crystallography and experimental single-wavelength anomalous dispersion (SAD) or multiple-wavelength anomalous dispersion (MAD) phasing based on initial data-processing statistics and sample crystal properties. Such a predictive tool can rapidly assess the usefulness of data and guide the collection of an optimal data set. The increase in data rates from modern macromolecular crystallography beamlines, together with a demand from users for real-time feedback, has led to pressure on computational resources and a need for smarter data handling. Statistical and machine-learning methods have been applied to construct a classifier that displays 95% accuracy for training and testing data sets compiled from 440 solved structures. Applying this classifier to new data achieved 79% accuracy. These scores already provide clear guidance as to the effective use of computing resources and offer a starting point for a personalized data-collection assistant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2052252520000895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055369PMC
March 2020

Correction to: Joint X-ray/NMR structure refinement of multidomain/multisubunit systems.

J Biomol NMR 2019 Jul;73(6-7):279

Magnetic Resonance Center (CERM) and Interuniversity Consortium for Magnetic Resonance of Metallo Proteins (CIRMMP), Via L. Sacconi 6, 50019, Sesto Fiorentino, Italy.

The article "Joint X-ray/NMR structure refinement of multidomain/multisubunit systems" written by "Azzurra Carlon, Enrico Ravera, Giacomo Parigi, Garib N. Murshudov and Claudio Luchinat" was originally published electronically on the publisher's internet portal (currently SpringerLink) on 11 October 2018 without open access.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10858-019-00238-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692445PMC
July 2019

Analysis and validation of macromolecular B values.

Acta Crystallogr D Struct Biol 2019 May 30;75(Pt 5):505-518. Epub 2019 Apr 30.

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

This paper describes a global analysis of macromolecular B values. It is shown that the distribution of B values generally follows the shifted inverse-gamma distribution (SIGD). The parameters of the SIGD are estimated using the Fisher scoring technique with the expected Fisher information matrix. It is demonstrated that a contour plot based on the parameters of the SIGD can play a role in the validation of macromolecular structures. The dependence of the peak-height distribution on resolution and atomic B values is also analysed. It is demonstrated that the B-value distribution can have a dramatically different effect on peak heights at different resolutions. Consequently, a comparative analysis of the B values of neighbouring atoms must account for resolution. A combination of the SIGD, peak-height distribution and outlier detection was used to identify a number of entries from the PDB that require attention. It is also shown that the presence of a multimodal B-value distribution often indicates that some loops or parts of the molecule have either been mismodelled or have dramatically different mobility, depending on their environment within the crystal. These distributions can also indicate the level of sharpening/blurring used before atomic structure refinement. It is recommended that procedures such as sharpening/blurring should be avoided during refinement, although they can play important roles in map visualization and model building.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798319004807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503761PMC
May 2019

Joint X-ray/NMR structure refinement of multidomain/multisubunit systems.

J Biomol NMR 2019 Jul 11;73(6-7):265-278. Epub 2018 Oct 11.

Magnetic Resonance Center (CERM) and Interuniversity Consortium for Magnetic Resonance of Metallo Proteins (CIRMMP), Via L. Sacconi 6, 50019, Sesto Fiorentino, Italy.

Data integration in structural biology has become a paradigm for the characterization of biomolecular systems, and it is now accepted that combining different techniques can fill the gaps in each other's blind spots. In this frame, one of the combinations, which we have implemented in REFMAC-NMR, is residual dipolar couplings from NMR together with experimental data from X-ray diffraction. The first are exquisitely sensitive to the local details but does not give any information about overall shape, whereas the latter encodes more the information about the overall shape but at the same time tends to miss the local details even at the highest resolutions. Once crystals are obtained, it is often rather easy to obtain a complete X-ray dataset, however it is time-consuming to obtain an exhaustive NMR dataset. Here, we discuss the effect of including a-priori knowledge on the properties of the system to reduce the number of experimental data needed to obtain a more complete picture. We thus introduce a set of new features of REFMAC-NMR that allow for improved handling of RDC data for multidomain proteins and multisubunit biomolecular complexes, and encompasses the use of pseudo-contact shifts as an additional source of NMR-based information. The new feature may either help in improving the refinement, or assist in spotting differences between the crystal and the solution data. We show three different examples where NMR and X-ray data can be reconciled to a unique structural model without invoking mobility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10858-018-0212-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692505PMC
July 2019

Pathological macromolecular crystallographic data affected by twinning, partial-disorder and exhibiting multiple lattices for testing of data processing and refinement tools.

Sci Rep 2018 10 5;8(1):14876. Epub 2018 Oct 5.

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Twinning is a crystal growth anomaly, which has posed a challenge in macromolecular crystallography (MX) since the earliest days. Many approaches have been used to treat twinned data in order to extract structural information. However, in most cases it is usually simpler to rescreen for new crystallization conditions that yield an untwinned crystal form or, if possible, collect data from non-twinned parts of the crystal. Here, we report 11 structures of engineered variants of the E. coli enzyme N-acetyl-neuraminic lyase which, despite twinning and incommensurate modulation, have been successfully indexed, solved and deposited. These structures span a resolution range of 1.45-2.30 Å, which is unusually high for datasets presenting such lattice disorders in MX and therefore these data provide an excellent test set for improving and challenging MX data processing programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-32962-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173773PMC
October 2018

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

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

Background modelling of diffraction data in the presence of ice rings.

IUCrJ 2017 Sep 8;4(Pt 5):626-638. Epub 2017 Aug 8.

Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.

An algorithm for modelling the background for each Bragg reflection in a series of X-ray diffraction images containing Debye-Scherrer diffraction from ice in the sample is presented. The method involves the use of a global background model which is generated from the complete X-ray diffraction data set. Fitting of this model to the background pixels is then performed for each reflection independently. The algorithm uses a static background model that does not vary over the course of the scan. The greatest improvement can be expected for data where ice rings are present throughout the data set and the local background shape at the size of a spot on the detector does not exhibit large time-dependent variation. However, the algorithm has been applied to data sets whose background showed large pixel variations (variance/mean > 2) and has been shown to improve the results of processing for these data sets. It is shown that the use of a simple flat-background model as in traditional integration programs causes systematic bias in the background determination at ice-ring resolutions, resulting in an overestimation of reflection intensities at the peaks of the ice rings and an underestimation of reflection intensities either side of the ice ring. The new global background-model algorithm presented here corrects for this bias, resulting in a noticeable improvement in factors following refinement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2052252517010259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619854PMC
September 2017

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

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

Robust background modelling in .

J Appl Crystallogr 2016 Dec 21;49(Pt 6):1912-1921. Epub 2016 Oct 21.

Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK.

A method for estimating the background under each reflection during integration that is robust in the presence of pixel outliers is presented. The method uses a generalized linear model approach that is more appropriate for use with Poisson distributed data than traditional approaches to pixel outlier handling in integration programs. The algorithm is most applicable to data with a very low background level where assumptions of a normal distribution are no longer valid as an approximation to the Poisson distribution. It is shown that traditional methods can result in the systematic underestimation of background values. This then results in the reflection intensities being overestimated and gives rise to a change in the overall distribution of reflection intensities in a dataset such that too few weak reflections appear to be recorded. Statistical tests performed during data reduction may mistakenly attribute this to merohedral twinning in the crystal. Application of the robust generalized linear model algorithm is shown to correct for this bias.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1600576716013595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139990PMC
December 2016

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

How to tackle protein structural data from solution and solid state: An integrated approach.

Prog Nucl Magn Reson Spectrosc 2016 Feb 21;92-93:54-70. Epub 2016 Jan 21.

Magnetic Resonance Center (CERM) and Department of Chemistry "Ugo Schiff", University of Florence, Italy(1). Electronic address:

Long-range NMR restraints, such as diamagnetic residual dipolar couplings and paramagnetic data, can be used to determine 3D structures of macromolecules. They are also used to monitor, and potentially to improve, the accuracy of a macromolecular structure in solution by validating or "correcting" a crystal model. Since crystal structures suffer from crystal packing forces they may not be accurate models for the macromolecular structures in solution. However, the presence of real differences should be tested for by simultaneous refinement of the structure using both crystal and solution NMR data. To achieve this, the program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic and paramagnetic NMR data and/or diamagnetic residual dipolar couplings. Inconsistencies between crystal structures and solution NMR data, if any, may be due either to structural rearrangements occurring on passing from the solution to solid state, or to a greater degree of conformational heterogeneity in solution with respect to the crystal. In the case of multidomain proteins, paramagnetic restraints can provide the correct mutual orientations and positions of domains in solution, as well as information on the conformational variability experienced by the macromolecule.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pnmrs.2016.01.001DOI Listing
February 2016

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

Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Nature 2015 Jul 27;523(7558):106-10. Epub 2015 Apr 27.

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

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature14356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493928PMC
July 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

The PDB_REDO server for macromolecular structure model optimization.

IUCrJ 2014 Jul 30;1(Pt 4):213-20. Epub 2014 May 30.

Division of Biochemistry, Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

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 crystallo-graphers 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2052252514009324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107921PMC
July 2014

Simultaneous use of solution NMR and X-ray data in REFMAC5 for joint refinement/detection of structural differences.

Acta Crystallogr D Biol Crystallogr 2014 Apr 19;70(Pt 4):958-67. Epub 2014 Mar 19.

Center for Magnetic Resonance (CERM), University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino (FI), Italy.

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004713034160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306559PMC
April 2014

High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy.

Ultramicroscopy 2013 Dec 21;135:24-35. Epub 2013 Jun 21.

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

Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultramic.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834153PMC
December 2013

How good are my data and what is the resolution?

Acta Crystallogr D Biol Crystallogr 2013 Jul 13;69(Pt 7):1204-14. Epub 2013 Jun 13.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, England.

Following integration of the observed diffraction spots, the process of `data reduction' initially aims to determine the point-group symmetry of the data and the likely space group. This can be performed with the program POINTLESS. The scaling program then puts all the measurements on a common scale, averages measurements of symmetry-related reflections (using the symmetry determined previously) and produces many statistics that provide the first important measures of data quality. A new scaling program, AIMLESS, implements scaling models similar to those in SCALA but adds some additional analyses. From the analyses, a number of decisions can be made about the quality of the data and whether some measurements should be discarded. The effective `resolution' of a data set is a difficult and possibly contentious question (particularly with referees of papers) and this is discussed in the light of tests comparing the data-processing statistics with trials of refinement against observed and simulated data, and automated model-building and comparison of maps calculated with different resolution limits. These trials show that adding weak high-resolution data beyond the commonly used limits may make some improvement and does no harm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0907444913000061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689523PMC
July 2013

PDB_REDO: constructive validation, more than just looking for errors.

Acta Crystallogr D Biol Crystallogr 2012 Apr 16;68(Pt 4):484-96. Epub 2012 Mar 16.

Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Developments of the PDB_REDO procedure that combine re-refinement and rebuilding within a unique decision-making framework to improve structures in the PDB are presented. PDB_REDO uses a variety of existing and custom-built software modules to choose an optimal refinement protocol (e.g. anisotropic, isotropic or overall B-factor refinement, TLS model) and to optimize the geometry versus data-refinement weights. Next, it proceeds to rebuild side chains and peptide planes before a final optimization round. PDB_REDO works fully automatically without the need for intervention by a crystallographic expert. The pipeline was tested on 12 000 PDB entries and the great majority of the test cases improved both in terms of crystallographic criteria such as R(free) and in terms of widely accepted geometric validation criteria. It is concluded that PDB_REDO is useful to update the otherwise `static' structures in the PDB to modern crystallographic standards. The publically available PDB_REDO database provides better model statistics and contributes to better refinement and validation targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0907444911054515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322608PMC
April 2012

JLigand: a graphical tool for the CCP4 template-restraint library.

Acta Crystallogr D Biol Crystallogr 2012 Apr 17;68(Pt 4):431-40. Epub 2012 Mar 17.

CCP4, STFC Rutherford Appleton Laboratory, Harwell Oxford, Didcot OX11 0QX, England.

Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user's additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user's library and generate new monomer entries is described, as well as new entries for links and associated modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S090744491200251XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322602PMC
April 2012

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

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

Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling.

J Biol Chem 2011 Jul 12;286(27):24208-18. Epub 2011 May 12.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1-4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.226142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129202PMC
July 2011