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
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

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, 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.

Metrics

  • 14,128
    views
  • 2,989
    downloads
  • 144
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Categories and tags

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.

    1. Structural Biology and Molecular Biophysics

    Cryo-EM: Protein complexes in focus

    Robert M Glaeser
    Insight

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

    1. Computational and Systems Biology
    2. Structural Biology and Molecular Biophysics

    Discovery of a heparan sulfate binding domain in monkeypox virus H3 as an anti-poxviral drug target combining AI and MD simulations

    Bin Zheng, Meimei Duan ... Peng Zheng
    Research Article

    Viral adhesion to host cells is a critical step in infection for many viruses, including monkeypox virus (MPXV). In MPXV, the H3 protein mediates viral adhesion through its interaction with heparan sulfate (HS), yet the structural details of this interaction have remained elusive. Using AI-based structural prediction tools and molecular dynamics (MD) simulations, we identified a novel, positively charged α-helical domain in H3 that is essential for HS binding. This conserved domain, found across orthopoxviruses, was experimentally validated and shown to be critical for viral adhesion, making it an ideal target for antiviral drug development. Targeting this domain, we designed a protein inhibitor, which disrupted the H3-HS interaction, inhibited viral infection in vitro and viral replication in vivo, offering a promising antiviral candidate. Our findings reveal a novel therapeutic target of MPXV, demonstrating the potential of combination of AI-driven methods and MD simulations to accelerate antiviral drug discovery.