Blog
About

27
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Advances in Single-Particle Electron Cryomicroscopy Structure Determination applied to Sub-tomogram Averaging

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Summary

          Recent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore some of these advances to improve structures determined using electron cryotomography (cryo-ET) and sub-tomogram averaging. We implement a new three-dimensional model for the contrast transfer function, and use this in a regularized likelihood optimization algorithm as implemented in the RELION program. Using direct electron detector data, we apply both single-particle analysis and sub-tomogram averaging to analyze radiation-induced movements of the specimen. As in single-particle cryo-EM, we find that significant sample movements occur during tomographic data acquisition, and that these movements are substantially reduced through the use of ultrastable gold substrates. We obtain a sub-nanometer resolution structure of the hepatitis B capsid, and show that reducing radiation-induced specimen movement may be central to attempts at further improving tomogram quality and resolution.

          Highlights

          • The Bayesian approach in RELION is extended to sub-tomogram averaging
          • A new 3D CTF and missing-wedge model for sub-tomogram averaging is proposed
          • Ultrastable gold supports reduce radiation-induced motion in tomography tilt series
          • Using the above, an 8 Å structure of hepatitis B capsid from cryo-ET is presented

          Abstract

          Bharat et al. have harnessed recent innovations in specimen preparation, data collection, and image processing from the field of cryo-EM single-particle analysis to improve electron cryotomography and sub-tomogram averaging.

          Related collections

          Most cited references 58

          • Record: found
          • Abstract: found
          • Article: not found

          RELION: Implementation of a Bayesian approach to cryo-EM structure determination

           Sjors Scheres (2012)
          RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Computer visualization of three-dimensional image data using IMOD.

            We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Automated electron microscope tomography using robust prediction of specimen movements.

               D Mastronarde (2005)
              A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.
                Bookmark

                Author and article information

                Affiliations
                [1 ]Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
                Author notes
                []Corresponding author tbharat@ 123456mrc-lmb.cam.ac.uk
                [∗∗ ]Corresponding author scheres@ 123456mrc-lmb.cam.ac.uk
                Contributors
                Journal
                Structure
                Structure
                Structure(London, England:1993)
                Cell Press
                0969-2126
                1878-4186
                01 September 2015
                01 September 2015
                : 23
                : 9
                : 1743-1753
                S0969-2126(15)00279-8
                10.1016/j.str.2015.06.026
                4559595
                26256537
                © 2015 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                Categories
                Resource

                Molecular biology

                Comments

                Comment on this article