Interpretation of very low resolution X-ray electron-density maps using core objects.
Ontology highlight
ABSTRACT:
SUBMITTER: Heuser P
PROVIDER: S-EPMC2703575 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Structures of multisubunit macromolecular machines are primarily determined either by electron microscopy (EM) or by X-ray crystallography. In many cases, a structure for a complex can be obtained at low resolution (at a coarse level of detail) with EM and at a higher resolution (with finer detail) by X-ray crystallography. The integration of these two structural techniques is becoming increasingly important for the generation of atomic models of macromolecular complexes. A low-resolution EM image can be a powerful tool for obtaining the 'phase' information that is missing from an X-ray crystallography experiment; however, integration of EM and X-ray diffraction data has been technically challenging. Here we present a step-by-step protocol that explains how low-resolution EM maps can be placed in the crystallographic unit cell by molecular replacement, and how initial phases computed from the placed EM density are extended to high resolution by averaging maps over noncrystallographic symmetry. As the resolution gap between EM and X-ray crystallography continues to narrow, the use of EM maps to help with X-ray crystal structure determination, as described in this protocol, will become increasingly effective.
| S-EPMC4620705 | biostudies-literature
Project description:This paper introduces ISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density maps. ISOLDE combines interactive molecular-dynamics flexible fitting with modern molecular-graphics visualization and established structural biology libraries to provide an immersive interface wherein the model constantly acts to maintain physically realistic conformations as the user interacts with it by directly tugging atoms with a mouse or haptic interface or applying/removing restraints. In addition, common validation tasks are accelerated and visualized in real time. Using the recently described 3.8 Å resolution cryo-EM structure of the eukaryotic minichromosome maintenance (MCM) helicase complex as a case study, it is demonstrated how ISOLDE can be used alongside other modern refinement tools to avoid common pitfalls of low-resolution modelling and improve the quality of the final model. A detailed analysis of changes between the initial and final model provides a somewhat sobering insight into the dangers of relying on a small number of validation metrics to judge the quality of a low-resolution model.
| S-EPMC6096486 | biostudies-literature
Project description:The structures of large macromolecular complexes in different functional states can be determined by cryo-electron microscopy, which yields electron density maps of low to intermediate resolutions. The maps can be combined with high-resolution atomic structures of components of the complex, to produce a model for the complex that is more accurate than the formal resolution of the map. To this end, methods have been developed to dock atomic models into density maps rigidly or flexibly, and to refine a docked model so as to optimize the fit of the atomic model into the map. We have developed a new refinement method called YUP.SCX. The electron density map is converted into a component of the potential energy function to which terms for stereochemical restraints and volume exclusion are added. The potential energy function is then minimized (using simulated annealing) to yield a stereochemically-restrained atomic structure that fits into the electron density map optimally. We used this procedure to construct an atomic model of the 70S ribosome in the pre-accommodation state. Although some atoms are displaced by as much as 33A, they divide themselves into nearly rigid fragments along natural boundaries with smooth transitions between the fragments.
| S-EPMC2702770 | biostudies-literature
Project description:Brickworx is a computer program that builds crystal structure models of nucleic acid molecules using recurrent motifs including double-stranded helices. In a first step, the program searches for electron-density peaks that may correspond to phosphate groups; it may also take into account phosphate-group positions provided by the user. Subsequently, comparing the three-dimensional patterns of the P atoms with a database of nucleic acid fragments, it finds the matching positions of the double-stranded helical motifs (A-RNA or B-DNA) in the unit cell. If the target structure is RNA, the helical fragments are further extended with recurrent RNA motifs from a fragment library that contains single-stranded segments. Finally, the matched motifs are merged and refined in real space to find the most likely conformations, including a fit of the sequence to the electron-density map. The Brickworx program is available for download and as a web server at http://iimcb.genesilico.pl/brickworx.
| S-EPMC4356372 | biostudies-literature
Project description:MotivationOwing to the size and complexity of large multi-component biological assemblies, the most tractable approach to determining their atomic structure is often to fit high-resolution radiographic or nuclear magnetic resonance structures of isolated components into lower resolution electron density maps of the larger assembly obtained using cryo-electron microscopy (cryo-EM). This hybrid approach to structure determination requires that an atomic resolution structure of each component, or a suitable homolog, is available. If neither is available, then the amount of structural information regarding that component is limited by the resolution of the cryo-EM map. However, even if a suitable homolog cannot be identified using sequence analysis, a search for structural homologs should still be performed because structural homology often persists throughout evolution even when sequence homology is undetectable, As macromolecules can often be described as a collection of independently folded domains, one way of searching for structural homologs would be to systematically fit representative domain structures from a protein domain database into the medium/low resolution cryo-EM map and return the best fits. Taken together, the best fitting non-overlapping structures would constitute a 'mosaic' backbone model of the assembly that could aid map interpretation and illuminate biological function.ResultUsing the computational principles of the Scale-Invariant Feature Transform (SIFT), we have developed FOLD-EM-a computational tool that can identify folded macromolecular domains in medium to low resolution (4-15 Å) electron density maps and return a model of the constituent polypeptides in a fully automated fashion. As a by-product, FOLD-EM can also do flexible multi-domain fitting that may provide insight into conformational changes that occur in macromolecular assemblies.
| S-EPMC3519459 | biostudies-literature
Project description:Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (cryo-ET) produce 3-D density maps of biological molecules at a range of resolution levels. Pattern recognition tools are important in distinguishing biological components from volumetric maps with the available resolutions. One of the most distinct characters in density maps at medium (5-10 Å) resolution is the visibility of protein secondary structures. Although computational methods have been developed, the accurate detection of helices and β-strands from cryo-EM density maps is still an active research area. We have developed a tool for protein secondary structure detection and evaluation of medium resolution 3-D cryo-EM density maps which combines three computational methods (SSETracer, StrandTwister, and AxisComparison). The program was integrated in UCSF Chimera, a popular visualization software in the cryo-EM community. In related work, we have developed BundleTrac, a computational method to trace filaments in a bundle from lower resolution cryo-ET density maps. It has been applied to actin filament tracing in stereocilia with good accuracy and can be potentially added as a tool in Chimera.
| S-EPMC9645795 | biostudies-literature
Project description:Cryo-electron microscopy of "single particles" is a powerful method to analyze structures of large macromolecular assemblies that are not amenable to investigation by traditional X-ray crystallographic methods. A key step in these studies is to obtain atomic interpretations of multiprotein complexes by fitting atomic structures of individual components into maps obtained from electron microscopic data. Here, we report the use of a "core-weighting" method, combined with a grid-threading Monte Carlo (GTMC) approach for this purpose. The "core" of an individual structure is defined to represent the part where the density distribution is least likely to be altered by other components that comprise the macromolecular assembly of interest. The performance of the method has been evaluated by its ability to determine the correct fit of (i) the alpha-chain of the T-cell receptor variable domain into a simulated map of the alphabeta complex at resolutions between 5 and 40 A, and (ii) the E2 catalytic domain of the pyruvate dehydrogenase into an experimentally determined map, at 14 A resolution, of the icosahedral complex formed by 60 copies of this enzyme. Using the X-ray structures of the two test cases as references, we demonstrate that, in contrast to more traditional methods, the combination of the core-weighting method and the grid-threading Monte Carlo approach can identify the correct fit reliably and rapidly from the low-resolution maps that are typical of structures determined with the use of single-particle electron microscopy.
| S-EPMC6413516 | biostudies-literature
Project description:High-quality three-dimensional structural data is of great value for the functional interpretation of biomacromolecules, especially proteins; however, structural quality varies greatly across the entries in the worldwide Protein Data Bank (wwPDB). Since 2008, the wwPDB has required the inclusion of structure factors with the deposition of x-ray crystallographic structures to support the independent evaluation of structures with respect to the underlying experimental data used to derive those structures. However, interpreting the discrepancies between the structural model and its underlying electron density data is difficult, since derived sigma-scaled electron density maps use arbitrary electron density units which are inconsistent between maps from different wwPDB entries. Therefore, we have developed a method that converts electron density values from sigma-scaled electron density maps into units of electrons. With this conversion, we have developed new methods that can evaluate specific regions of an x-ray crystallographic structure with respect to a physicochemical interpretation of its corresponding electron density map. We have systematically compared all deposited x-ray crystallographic protein models in the wwPDB with their underlying electron density maps, if available, and characterized the electron density in terms of expected numbers of electrons based on the structural model. The methods generated coherent evaluation metrics throughout all PDB entries with associated electron density data, which are consistent with visualization software that would normally be used for manual quality assessment. To our knowledge, this is the first attempt to derive units of electrons directly from electron density maps without the aid of the underlying structure factors. These new metrics are biochemically-informative and can be extremely useful for filtering out low-quality structural regions from inclusion into systematic analyses that span large numbers of PDB entries. Furthermore, these new metrics will improve the ability of non-crystallographers to evaluate regions of interest within PDB entries, since only the PDB structure and the associated electron density maps are needed. These new methods are available as a well-documented Python package on GitHub and the Python Package Index under a modified Clear BSD open source license.
| S-EPMC7423092 | biostudies-literature
Project description:Solution scattering techniques, such as small and wide-angle X-ray scattering (SWAXS), provide valuable insights into the structure and dynamics of biological macromolecules in solution. In this study, we present an approach to accurately predict solution X-ray scattering profiles at wide angles from atomic models by generating high-resolution electron density maps. Our method accounts for the excluded volume of bulk solvent by calculating unique adjusted atomic volumes directly from the atomic coordinates. This approach eliminates the need for a free fitting parameter commonly used in existing algorithms, resulting in improved accuracy of the calculated SWAXS profile. An implicit model of the hydration shell is generated which uses the form factor of water. Two parameters, namely the bulk solvent density and the mean hydration shell contrast, are adjusted to best fit the data. Results using eight publicly available SWAXS profiles show high quality fits to the data. In each case, the optimized parameter values show small adjustments demonstrating that the default values are close to the true solution. Disabling parameter optimization results in a significant improvement of the calculated scattering profiles compared to the leading software. The algorithm is computationally efficient, showing more than tenfold reduction in execution time compared to the leading software. The algorithm is encoded in a command line script called denss.pdb2mrc.py and is available open source as part of the DENSS v1.7.0 software package (https://github.com/tdgrant1/denss). In addition to improving the ability to compare atomic models to experimental SWAXS data, these developments pave the way for increasing the accuracy of modeling algorithms utilizing SWAXS data while decreasing the risk of overfitting.
| S-EPMC10312546 | biostudies-literature
Project description:Cryo-electron microscopy (cryo-EM) density maps at medium resolution (5-10 Å) reveal secondary structural features such as α-helices and β-sheets, but they lack the side chain details that would enable a direct structure determination. Among the more than 800 entries in the Electron Microscopy Data Bank (EMDB) of medium-resolution density maps that are associated with atomic models, a wide variety of similarities can be observed between maps and models. To validate such atomic models and to classify structural features, a local similarity criterion, the F1 score, is proposed and evaluated in this study. The F1 score is theoretically normalized to a range from zero to one, providing a local measure of cylindrical agreement between the density and atomic model of a helix. A systematic scan of 30,994 helices (among 3,247 protein chains modeled into medium-resolution density maps) reveals an actual range of observed F1 scores from 0.171 to 0.848, suggesting that the cylindrical fit of the current data is well stratified by the proposed measure. The best (highest) F1 scores tend to be associated with regions that exhibit high and spatially homogeneous local resolution (between 5 Å and 7.5 Å) in the helical density. The proposed F1 scores can be used as a discriminative classifier for validation studies and as a ranking criterion for cryo-EM density features in databases.
| S-EPMC8279803 | biostudies-literature