Routine single particle CryoEM sample and grid characterization by tomography.

Published online

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Noble, AJ; Dandey, VP; Wei, H; Brasch, J; Chase, J; Acharya, P; Tan, YZ; Zhang, Z; Kim, LY; Scapin, G; Rapp, M; Eng, ET; Rice, WJ; Cheng, A; Negro, CJ; Shapiro, L; Kwong, PD; Jeruzalmi, D; des Georges, A; Potter, CS; Carragher, B

Published Date

  • May 29, 2018

Published In

Volume / Issue

  • 7 /

PubMed ID

  • 29809143

Pubmed Central ID

  • 29809143

Electronic International Standard Serial Number (EISSN)

  • 2050-084X

Digital Object Identifier (DOI)

  • 10.7554/eLife.34257

Language

  • eng

Conference Location

  • England