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Routine single particle CryoEM sample and grid characterization by tomography.

Alex J Noble | Venkata P Dandey | Hui Wei | Julia Brasch | Jillian Chase | Priyamvada Acharya | Yong Zi Tan | Zhening Zhang | Laura Y Kim | Giovanna Scapin | Micah Rapp | Edward T Eng | William J Rice | Anchi Cheng | Carl J Negro | Lawrence Shapiro | Peter D Kwong | David Jeruzalmi | Amedee des Georges | Clinton S Potter | Bridget Carragher
eLife | 2018

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.

Pubmed ID: 29809143

Research resources used in this publication

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Antibodies used in this publication

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Associated grants

  • Agency: NIGMS NIH HHS, United States
    Id: P41 GM103310
  • Agency: NIH HHS, United States
    Id: S10 OD019994-01
  • Agency: NIMHD NIH HHS, United States
    Id: G12 MD007603
  • Agency: NIH HHS, United States
    Id: R01-MH1148175
  • Agency: NIAID NIH HHS, United States
    Id: Intramural Funding from the Vaccine Research Center
  • Agency: NIH HHS, United States
    Id: R01 GM084162
  • Agency: NIMHD NIH HHS, United States
    Id: 5G12MD007603-30
  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM084162
  • Agency: NIGMS NIH HHS, United States
    Id: GM103310
  • Agency: NIH HHS, United States
    Id: S10 OD019994

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This is a list of tools and resources that we have found mentioned in this publication.


IMOD (tool)

RRID:SCR_003297

A free, cross-platform set of image processing, modeling and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections. The package contains tools for assembling and aligning data within multiple types and sizes of image stacks, viewing 3-D data from any orientation, and modeling and display of the image files. IMOD 4.1.8 Is Now Available for Linux, Windows, and Mac OS X

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Electron Microscopy Public Image Archive (tool)

RRID:SCR_019237

Public archive for raw electron microscopy image data. Resource for raw 2D electron microscopy images. Used to browse, upload and download raw images to build 3D structure. Provides access to raw data to facilitate methods development and validation. Complements Electron Microscopy Data Bank where 3D volumes are stored, and uses Aspera platform for data transfers.

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