Cryo-EM single particle analysis with the Volta phase plate

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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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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"".
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.

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