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Elife. 2018 May 29;7. pii: e34257. doi: 10.7554/eLife.34257.

Routine single particle CryoEM sample and grid characterization by tomography.

Author information

1
National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States.
2
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States.
3
Department of Chemistry and Biochemistry, City College of New York, New York, United States.
4
Program in Biochemistry, The Graduate Center of the City University of New York, New York, United States.
5
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Maryland, United States.
6
Department of Structural Chemistry and Chemical Biotechnology, Merck & Co., Inc, New Jersey, United States.
7
Program in Biology, The Graduate Center of the City University of New York, New York, United States.
8
Program in Chemistry, The Graduate Center of the City University of New York, New York, United States.
9
Advanced Science Research Center, The Graduate Center of the City University of New York, New York, United States.
#
Contributed equally

Abstract

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

KEYWORDS:

air-water; cryoET; fiducial-less; molecular biophysics; none; protomo; single particle; structural biology; tomography

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