Routine single particle CryoEM sample and grid characterization by tomography

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Air / analysis
  • Animals
  • Apoferritins / ultrastructure
  • Cryoelectron Microscopy / instrumentation*
  • Cryoelectron Microscopy / methods
  • DnaB Helicases / ultrastructure
  • Electron Microscope Tomography / instrumentation*
  • Electron Microscope Tomography / methods
  • Escherichia coli / chemistry
  • Escherichia coli / enzymology
  • Fructose-Bisphosphate Aldolase / ultrastructure
  • Proteasome Endopeptidase Complex / ultrastructure
  • Rabbits
  • Sugar Alcohol Dehydrogenases / ultrastructure
  • Surface Properties
  • Water / chemistry

Substances

  • Water
  • Apoferritins
  • Sugar Alcohol Dehydrogenases
  • galactitol 2-dehydrogenase
  • Proteasome Endopeptidase Complex
  • dnaB protein, E coli
  • DnaB Helicases
  • Fructose-Bisphosphate Aldolase