1. Structural Biology and Molecular Biophysics

Using the Volta phase plate with defocus for cryo-EM single particle analysis

  1. Radostin Danev  Is a corresponding author
  2. Dimitry Tegunov
  3. Wolfgang Baumeister
  1. Max Planck Institute of Biochemistry, Germany
  2. Max Planck Institute for Biophysical Chemistry, Germany
https://doi.org/10.7554/eLife.23006
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  1. Radostin Danev
  2. Dimitry Tegunov
  3. Wolfgang Baumeister
(2017)
Using the Volta phase plate with defocus for cryo-EM single particle analysis
eLife 6:e23006.
https://doi.org/10.7554/eLife.23006
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Abstract

Previously, we reported an in-focus data acquisition method for cryo-EM single particle analysis with the Volta phase plate (VPP) (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.

Data availability

The following data sets were generated
    1. Danev R
    2. Tegunov D
    3. Baumeister W
    (2016) Volta phase plate with defocus cryo-EM dataset of Thermoplasma acidophilum 20S proteasome
    Publicly available at the Electron Microscopy Pilot Image Archive (accession no: EMPIAR-10078).
    https://www.ebi.ac.uk/pdbe/emdb/empiar/

Article and author information

Author details

  1. Radostin Danev

    Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    For correspondence
    danev@biochem.mpg.de
    Competing interests
    Radostin Danev, RD is a co-inventor in US patent US9129774 B2 Method of using a phase plate in a transmission electron microscope"".
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6406-8993

  2. Dimitry Tegunov

    Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7019-3221

  3. Wolfgang Baumeister

    Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    Wolfgang Baumeister, WB is on the Scientific Advisory Board of FEI Company.

Funding

Max-Planck-Gesellschaft

  • Radostin Danev
  • Dimitry Tegunov
  • Wolfgang Baumeister

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

Reviewing Editor

  1. Sjors HW Scheres, MRC Laboratory of Molecular Biology,, United Kingdom

Version history

  1. Received: November 7, 2016
  2. Accepted: January 20, 2017
  3. Accepted Manuscript published: January 21, 2017 (version 1)
  4. Version of Record published: January 30, 2017 (version 2)

Copyright

© 2017, Danev 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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  1. Radostin Danev
  2. Dimitry Tegunov
  3. Wolfgang Baumeister
(2017)
Using the Volta phase plate with defocus for cryo-EM single particle analysis
eLife 6:e23006.
https://doi.org/10.7554/eLife.23006

Share this article

https://doi.org/10.7554/eLife.23006

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Further reading

    1. Structural Biology and Molecular Biophysics

    Cryo-EM single particle analysis with the Volta phase plate

    Radostin Danev, Wolfgang Baumeister
    Tools and Resources Updated

    We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.

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