1. Structural Biology and Molecular Biophysics

Cryo-EM single particle analysis with the Volta phase plate

  1. Radostin Danev  Is a corresponding author
  2. Wolfgang Baumeister
  1. Max Planck Institute of Biochemistry, Germany
https://doi.org/10.7554/eLife.13046
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  1. Radostin Danev
  2. Wolfgang Baumeister
(2016)
Cryo-EM single particle analysis with the Volta phase plate
eLife 5:e13046.
https://doi.org/10.7554/eLife.13046
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Abstract

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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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, co-inventor in US patent US9129774 B2 Method of using a phase plate in a transmission electron microscope"".

  2. Wolfgang Baumeister

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

Copyright

© 2016, Danev & Baumeister

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. Wolfgang Baumeister
(2016)
Cryo-EM single particle analysis with the Volta phase plate
eLife 5:e13046.
https://doi.org/10.7554/eLife.13046

Share this article

https://doi.org/10.7554/eLife.13046

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Further reading

    1. Structural Biology and Molecular Biophysics

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

    Radostin Danev, Dimitry Tegunov, Wolfgang Baumeister
    Research Advance Updated

    Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (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.

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    Cryo-EM: Protein complexes in focus

    Robert M Glaeser
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    A new advance in electron microscopy can reveal highly-detailed structures of protein complexes.

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