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      An atomic model of brome mosaic virus using direct electron detection and real-space optimization

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          Abstract

          Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

          Abstract

          Recent developments in cryo-electron microscopy have enabled structure determination of large protein complexes at almost atomic resolution. Wang et al. combine some of these technologies into an effective workflow, and demonstrate the protocol by solving the atomic structure of an icosahedral RNA virus.

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          Most cited references25

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          Improved methods for building protein models in electron density maps and the location of errors in these models.

          Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
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            Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles

            Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens. DOI: http://dx.doi.org/10.7554/eLife.00461.001
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              Movies of ice-embedded particles enhance resolution in electron cryo-microscopy.

              Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence. Copyright © 2012 Elsevier Ltd. All rights reserved.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                04 September 2014
                : 5
                : 4808
                Affiliations
                [1 ]National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine , Houston, Texas 77030, USA
                [2 ]Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine , Houston, Texas 77030, USA
                [3 ]Direct Electron LP , San Diego, California 92128, USA
                [4 ]Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, USA
                [5 ]Department of Molecular & Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, USA
                [6 ]Department of Bioengineering, University of California , Berkeley, California 94720, USA
                [7 ]These authors contributed equally to this work
                Author notes
                Article
                ncomms5808
                10.1038/ncomms5808
                4155512
                25185801
                b2eee85d-a31f-47c4-bf85-df750564310c
                Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

                History
                : 12 March 2014
                : 24 July 2014
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