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J Struct Biol. 2018 Nov;204(2):270-275. doi: 10.1016/j.jsb.2018.07.015. Epub 2018 Jul 25.

High resolution single particle cryo-electron microscopy using beam-image shift.

Author information

1
Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA; National Resource for Automated Molecular Microscopy, New York Structural Biology Center, New York, NY 10027, USA. Electronic address: acheng@nysbc.org.
2
Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA.
3
Thermo Fisher Scientific, Materials & Structural Analysis, Hillsboro, OR 97124, USA.
4
Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA; National Resource for Automated Molecular Microscopy, New York Structural Biology Center, New York, NY 10027, USA.

Abstract

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

KEYWORDS:

Automation; Coma free alignment; Cryo-EM; Phase error

PMID:
30055234
PMCID:
PMC6163078
[Available on 2019-11-01]
DOI:
10.1016/j.jsb.2018.07.015

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