1. Structural Biology and Molecular Biophysics
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Locating Macromolecular Assemblies in Cells by 2D Template Matching with cisTEM

  1. Bronwyn A Lucas
  2. Benjamin A Himes
  3. Liang Xue
  4. Tim Grant
  5. Julia Mahamid
  6. Nikolaus Grigorieff  Is a corresponding author
  1. Howard Hughes Medical Institute, United States
  2. University of Massachusetts Medical School, United States
  3. EMBL, Germany
  4. European Molecular Biology Laboratory, Germany
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Cite this article as: eLife 2021;10:e68946 doi: 10.7554/eLife.68946

Abstract

For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer resolution in three dimensions (3D) using electron cryo-tomography, which requires tilt series to be recorded and computationally aligned, currently limiting throughput. Additionally, the high-resolution signal preserved in the raw tomograms is currently limited by a number of technical difficulties, leading to an increased false-positive detection rate when using 3D template matching to find molecular complexes in tomograms. We have recently described a 2D template matching approach that addresses these issues by including high-resolution signal preserved in single-tilt images. A current limitation of this approach is the high computational cost that limits throughput. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells with high precision and sensitivity, demonstrating that this is a versatile tool for in situ visual proteomics and in situ structure determination. We benchmark the results with 3D template matching of tomograms acquired on identical sample locations and identify strengths and weaknesses of both techniques, which offer complementary information about target localization and identity.

Data availability

All the code used for the 2D template matching has an open source license and is freely available from the cisTEM github repository, https://github.com/timothygrant80/cisTEM. The images of M. pneumoniae analyzed for this work, as well as the 70S reconstructions have been deposited in the EMPIAR and EMDB databases, https://www.ebi.ac.uk/pdbe/emdb/empiar/ and https://www.ebi.ac.uk/pdbe/emdb,

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Article and author information

Author details

  1. Bronwyn A Lucas

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.
  2. Benjamin A Himes

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7777-0298
  3. Liang Xue

    Structural and Computational Biology Unit, EMBL, Heidelberg, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4368-2526
  4. Tim Grant

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4855-8703
  5. Julia Mahamid

    European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6968-041X
  6. Nikolaus Grigorieff

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    niko@grigorieff.org
    Competing interests
    Nikolaus Grigorieff, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X

Funding

European Research Council (760067)

  • Julia Mahamid

Howard Hughes Medical Institute (N/A)

  • Bronwyn A Lucas
  • Benjamin A Himes
  • Nikolaus Grigorieff

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Edward H Egelman, University of Virginia, United States

Publication history

  1. Received: March 30, 2021
  2. Accepted: June 9, 2021
  3. Accepted Manuscript published: June 11, 2021 (version 1)

Copyright

© 2021, Lucas et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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