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J Struct Biol. 2018 Oct;204(1):38-44. doi: 10.1016/j.jsb.2018.06.007. Epub 2018 Jul 4.

Routine determination of ice thickness for cryo-EM grids.

Author information

1
National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States.
2
National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States.

Abstract

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

KEYWORDS:

Cryo-EM; Energy filter; Ice thickness; Inelastic scattering; Mean free path

PMID:
29981485
PMCID:
PMC6119488
[Available on 2019-10-01]
DOI:
10.1016/j.jsb.2018.06.007

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