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J Microsc. 2019 Oct;276(1):39-45. doi: 10.1111/jmi.12834. Epub 2019 Oct 7.

Current outcomes when optimizing 'standard' sample preparation for single-particle cryo-EM.

Author information

1
Simon Electron Microscopy Center, New York Structural Biology Center, New York, U.S.A.
2
HHMI and Department of Biochemistry and Biophysics, University of California, San Francisco, California, U.S.A.
3
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, U.S.A.
4
Lawrence Berkeley National Laboratory, University of California, Berkeley, California, U.S.A.
5
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, U.S.A.
6
Molecular and Cell Biology Department, University of California Berkeley, Berkeley, California, U.S.A.
7
MBIB Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California, U.S.A.
8
Howard Hughes Medical Institute, University of California Berkeley, Berkeley, California, U.S.A.
9
Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center of Biological Structures, School of Life Sciences, Tsinghua University, Beijing, China.

Abstract

Although high-resolution single-particle cryo-electron microscopy (cryo-EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the preparation of suitable frozen-hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous - while still in the test tube - may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo-EM field.

KEYWORDS:

Air-water interface; biological cryo-EM; sample preparation

PMID:
31553060
DOI:
10.1111/jmi.12834

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