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

Radostin Danev,Dimitry Tegunov,Wolfgang Baumeister

doi:10.7554/elife.23006

Radostin Danev, Dimitry Tegunov + Show 1 more

Open Access

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 (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.

Highlights

The Volta phase plate (VPP) is a device which enables in-focus phase contrast in a transmission electron microscope (TEM) (Danev et al, 2014)
Heterogeneous and/ or flexible molecules are generally difficult to solve by the traditional defocus phase contrast (DPC) method (Cheng et al, 2015)
The VPP provides low frequency contrast which allows the use of defocus values smaller than

Summary

Introduction

The Volta phase plate (VPP) is a device which enables in-focus phase contrast in a transmission electron microscope (TEM) (Danev et al, 2014). At the time of our initial report the 3D reconstruction software did not yet support VPP phase shift (PS) in the contrast transfer function (CTF). It simplifies the data acquisition to a level similar to DPC and solves the resolution limit issue of the in-focus approach by enabling CTF fitting and correction.

Results

Conclusion