New tools for the analysis and validation of cryo-EM maps and atomic models.
Ontology highlight
ABSTRACT:
SUBMITTER: Afonine PV
PROVIDER: S-EPMC6130467 | biostudies-literature | 2018 Sep
REPOSITORIES: biostudies-literature
Similar Datasets
Project description: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.
| S-EPMC6096485 | biostudies-literature
Project description:Although electron cryo-microscopy (cryo-EM) has recently achieved resolutions of better than 3 Å, at which point molecular modeling can be done directly from the density map, analysis and annotation of a cryo-EM density map still primarily rely on fitting atomic or homology models to the density map. In this article, we present, to our knowledge, a new method for flexible fitting of known or modeled protein structures into cryo-EM density maps. Unlike existing methods that are guided by local density gradients, our method is guided by correspondences between the ?-helices in the density map and model, and does not require an initial rigid-body fitting step. Compared with current methods on both simulated and experimental density maps, our method not only achieves greater accuracy for proteins with large deformations but also runs as fast or faster than many of the other flexible fitting routines.
| S-EPMC5479111 | biostudies-literature
Project description:BACKGROUND:Dramatic progress has recently been made in cryo-electron microscopy technologies, which now make possible the reconstruction of a growing number of biomolecular structures to near-atomic resolution. However, the need persists for fitting and refinement approaches that address those cases that require modeling assistance. METHODS:In this paper, we describe algorithms to optimize the performance of such medium-resolution refinement methods. These algorithms aim to automatically optimize the parameters that define the density shape of the flexibly fitted model, as well as the time-dependent damper cutoff distance. Atomic distance constraints can be prescribed for cases where extra containment of parts of the structure is helpful, such as in regions where the density map is poorly defined. Also, we propose a simple stopping criterion that estimates the probable onset of overfitting during the simulation. RESULTS:The new set of algorithms produce more accurate fitting and refinement results, and yield a faster rate of convergence of the trajectory toward the fitted conformation. The latter is also more reliable due to the overfitting warning provided to the user. CONCLUSIONS:The algorithms described here were implemented in the new Damped-Dynamics Flexible Fitting simulation tool "DDforge" in the Situs package.
| S-EPMC6139150 | biostudies-literature
Project description:We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-Å resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps.
| S-EPMC4435692 | biostudies-literature
Project description:Cryogenic electron microscopy (cryo-EM) is a powerful technique for determining structures of multiple conformational or compositional states of macromolecular assemblies involved in cellular processes. Recent technological developments have led to a leap in the resolution of many cryo-EM data sets, making atomic model building more common for data interpretation. We present a method for calculating differences between two cryo-EM maps or a map and a fitted atomic model. The proposed approach works by scaling the maps using amplitude matching in resolution shells. To account for variability in local resolution of cryo-EM data, we include a procedure for local amplitude scaling that enables appropriate scaling of local map contrast. The approach is implemented as a user-friendly tool in the CCP-EM software package. To obtain clean and interpretable differences, we propose a protocol involving steps to process the input maps and output differences. We demonstrate the utility of the method for identifying conformational and compositional differences including ligands. We also highlight the use of difference maps for evaluating atomic model fit in cryo-EM maps.
| S-EPMC7254831 | biostudies-literature
Project description:The appearance of ten high-resolution cryoelectron microscopy (cryo-EM) maps of proteins, ribosomes, and viruses was compared with the experimentally phased crystallographic electron density maps of four proteins. We found that maps calculated at a similar resolution by the two techniques are quite comparable in their appearance, although cryo-EM maps, even when sharpened, seem to be a little less detailed. An analysis of models fitted to the cryo-EM maps indicated the presence of significant problems in almost all of them, including incorrect geometry, clashes between atoms, and discrepancies between the map density and the fitted models. In particular, the treatment of the atomic displacement (B) factors was meaningless in almost all analyzed cryo-EM models. Stricter cryo-EM structure deposition standards and their better enforcement are needed.
| S-EPMC5657611 | biostudies-literature
Project description:We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a variable-metric gradient descent algorithm with adaptive moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on 12 publicly available cryo-EM data sets.
| S-EPMC8786306 | biostudies-literature
Project description:Confidence maps provide complementary information for interpreting cryo-EM densities as they indicate statistical significance with respect to background noise. They can be thresholded by specifying the expected false-discovery rate (FDR), and the displayed volume shows the parts of the map that have the corresponding level of significance. Here, the basic statistical concepts of confidence maps are reviewed and practical guidance is provided for their interpretation and usage inside the CCP-EM suite. Limitations of the approach are discussed and extensions towards other error criteria such as the family-wise error rate are presented. The observed map features can be rendered at a common isosurface threshold, which is particularly beneficial for the interpretation of weak and noisy densities. In the current article, a practical guide is provided to the recommended usage of confidence maps.
| S-EPMC7137106 | biostudies-literature
Project description:The rapid progress of cryo-electron microscopy (cryo-EM) in structural biology has raised an urgent need for robust methods to create and refine atomic-level structural models using low-resolution EM density maps. We propose a new protocol to create initial models using I-TASSER protein structure prediction, followed by EM density map-based rigid-body structure fitting, flexible fragment adjustment and atomic-level structure refinement simulations. The protocol was tested on a large set of 285 non-homologous proteins and generated structural models with correct folds for 260 proteins, where 28% had RMSDs below 2 Å. Compared to other state-of-the-art methods, the major advantage of the proposed pipeline lies in the uniform structure prediction and refinement protocol, as well as the extensive structural re-assembly simulations, which allow for low-to-medium resolution EM density map-guided structure modeling starting from amino acid sequences. Interestingly, the quality of both the image fitting and subsequent structure refinement was found to be strongly correlated with the correctness of the initial I-TASSER models; this is mainly due to the different correlation patterns observed between force field and structural quality for the models with template modeling score (or TM-score, a metric quantifying the similarity of models to the native) above and below a threshold of 0.5. Overall, the results demonstrate a new avenue that is ready to use for large-scale cryo-EM-based structure modeling and atomic-level density map-guided structure refinement.
| S-EPMC7502543 | biostudies-literature
Project description:Density modification uses expectations about features of a map such as a flat solvent and expected distributions of density in the region of the macromolecule to improve individual Fourier terms representing the map. This process transfers information from one part of a map to another and can improve the accuracy of a map. Here, the assumptions behind density modification for maps from electron cryomicroscopy are examined and a procedure is presented that allows the incorporation of model-based information. Density modification works best in cases where unfiltered, unmasked maps with clear boundaries between the macromolecule and solvent are visible, and where there is substantial noise in the map, both in the region of the macromolecule and the solvent. It also is most effective if the characteristics of the map are relatively constant within regions of the macromolecule and the solvent. Model-based information can be used to improve density modification, but model bias can in principle occur. Here, model bias is reduced by using ensemble models that allow an estimation of model uncertainty. A test of model bias is presented that suggests that even if the expected density in a region of a map is specified incorrectly by using an incorrect model, the incorrect expectations do not strongly affect the final map.
| S-EPMC7543659 | biostudies-literature