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

Abstract
Data availability
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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

(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/
(2016) Thermoplasma acidophilum 20S proteasome map reconstructed with Relion 2.0 particle polishing from defocused Volta phase plate cryo-EM data
Publicly available at the EMDB Protein Data Bank in Europe (accession no: EMD-3455).

https://www.ebi.ac.uk/pdbe/emdb/
(2016) Thermoplasma acidophilum 20S proteasome map reconstructed with MotionCor2 and Relion 2.0 from defocused Volta phase plate cryo-EM data
Publicly available at the EMDB Protein Data Bank in Europe (accession no: EMD-3456).

https://www.ebi.ac.uk/pdbe/emdb/

Article and author information

Author details

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"".

"This ORCID iD identifies the author of this article:" 0000-0001-6406-8993
Dimitry Tegunov

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-7019-3221
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.

Copyright

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.