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.
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.
Here, a macromolecule‐centred approach to three‐dimensional structure determination as implemented in REFMAC5 is considered. The use of restraints to transfer chemical and structural information during macromolecular refinement, and how different sources of information can be combined in order to achieve models that are more consistent with data derived from a variety of experimental techniques, including macromolecular crystallography, cryo‐EM and NMR spectroscopy, are discussed.
AceDRG: a stereochemical description generator for ligands Long, Fei; Nicholls, Robert A.; Emsley, Paul ...
Acta crystallographica. Section D, Structural biology,
February 2017, 2017-02-01, 20170201, Letnik:
73, Številka:
2
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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.
The program AceDRG generates accurate stereochemical descriptions, and one or more conformations, of a given ligand. The program also analyses entries and extracts local environment‐dependent atom types, bonds and angles from the Crystallography Open Database.
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.
REFMAC5 and related tools for the refinement of atomic models into cryo‐EM reconstructions in CCP‐EM are reviewed. An upper bound on the correlation between observed and calculated Fourier coefficients is identified, and the practical utility of map blurring/sharpening is discussed. The Divide and Conquer pipeline for refining large complexes in parallel, and ProSHADE for the identification of symmetries in a given map or coordinate set, are presented.
Low-resolution refinement tools in REFMAC5 Nicholls, Robert A.; Long, Fei; Murshudov, Garib N.
Acta crystallographica. Section D, Biological crystallography.,
April 2012, Letnik:
68, Številka:
4
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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.
To appraise the clinical and genetic evidence that low-density lipoproteins (LDLs) cause atherosclerotic cardiovascular disease (ASCVD).
We assessed whether the association between LDL and ASCVD ...fulfils the criteria for causality by evaluating the totality of evidence from genetic studies, prospective epidemiologic cohort studies, Mendelian randomization studies, and randomized trials of LDL-lowering therapies. In clinical studies, plasma LDL burden is usually estimated by determination of plasma LDL cholesterol level (LDL-C). Rare genetic mutations that cause reduced LDL receptor function lead to markedly higher LDL-C and a dose-dependent increase in the risk of ASCVD, whereas rare variants leading to lower LDL-C are associated with a correspondingly lower risk of ASCVD. Separate meta-analyses of over 200 prospective cohort studies, Mendelian randomization studies, and randomized trials including more than 2 million participants with over 20 million person-years of follow-up and over 150 000 cardiovascular events demonstrate a remarkably consistent dose-dependent log-linear association between the absolute magnitude of exposure of the vasculature to LDL-C and the risk of ASCVD; and this effect appears to increase with increasing duration of exposure to LDL-C. Both the naturally randomized genetic studies and the randomized intervention trials consistently demonstrate that any mechanism of lowering plasma LDL particle concentration should reduce the risk of ASCVD events proportional to the absolute reduction in LDL-C and the cumulative duration of exposure to lower LDL-C, provided that the achieved reduction in LDL-C is concordant with the reduction in LDL particle number and that there are no competing deleterious off-target effects.
Consistent evidence from numerous and multiple different types of clinical and genetic studies unequivocally establishes that LDL causes ASCVD.
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.
Ligand fitting with CCP4 Nicholls, Robert A.
Acta crystallographica. Section D, Structural biology,
February 2017, 2017-02-01, 20170201, Letnik:
73, Številka:
2
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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.
The process of ligand fitting with CCP4 is reviewed, including identifying ligand density in the map, ligand fitting, refinement and subsequent validation. Recent developments are discussed, and are illustrated using instructive examples demonstrating practical application.
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.
An automated pipeline for low‐resolution structure refinement (LORESTR) has been developed to assist in the hassle‐free refinement of difficult cases. The pipeline automates the selection of high‐resolution homologues for external restraint generation and optimizes the parameters for ProSMART and REFMAC5, improving R factors and geometry statistics in 94% of the test cases.
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.
CCP4i2 is a graphical user interface to the CCP4 (Collaborative Computational Project, Number 4) software suite and a Python language framework for software automation.
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