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 1,000 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.

Data availability

Several representative tilt-series from the datasets have been deposited to the Electron Microscopy Data Bank (EMDB) in the form of binned by 4 or 8 tomograms and to the Electron Microscopy Pilot Image Archive (EMPIAR) in the form of unaligned tilt-series images (one including super-resolution frames), Appion-Protomo tilt-series alignment runs, and aligned tilt-series stacks.Protomo estimations for the orientation of the local ice normal based on the tilt-series alignment of the particles in the ice, which includes potential systematic stage and beam axis error, are available in all deposited EMPIAR datasets as a plot located: protomo_alignments/tiltseries####/media/angle_refinement/series####_orientation.gifA Docker-based version of Appion-Protomo fiducial-less tilt-series alignment is available at http://github.com/nysbc/appion-protomo.

The following data sets were generated

Article and author information

Author details

  1. Alex J Noble

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8634-2279
  2. Venkata P Dandey

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Hui Wei

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Julia Brasch

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Jillian Chase

    Department of Chemistry and Biochemistry, City College of New York, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Priyamvada Acharya

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Yong Zi Tan

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6656-6320
  8. Zhening Zhang

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Laura Y Kim

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Giovanna Scapin

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Micah Rapp

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Edward T Eng

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8014-7269
  13. William J Rice

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Anchi Cheng

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Carl J Negro

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Lawrence Shapiro

    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Peter D Kwong

    Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. David Jeruzalmi

    Department of Chemistry and Biochemistry, City College of New York, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5886-1370
  19. Amedee des Georges

    Department of Chemistry and Biochemistry, City College of New York, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  20. Clinton S Potter

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2394-0831
  21. Bridget Carragher

    National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    For correspondence
    bcarr@nysbc.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0624-5020

Funding

Simons Foundation (SF349247)

  • Clinton S Potter
  • Bridget Carragher

National Institutes of Health (R01 GM084162)

  • David Jeruzalmi

New York State Foundation for Science, Technology and Innovation

  • Clinton S Potter
  • Bridget Carragher

National Institute of General Medical Sciences (GM103310)

  • Clinton S Potter
  • Bridget Carragher

Agouron Institute (F00316)

  • Clinton S Potter
  • Bridget Carragher

National Institutes of Health (S10 OD019994-01)

  • Clinton S Potter
  • Bridget Carragher

National Institute on Minority Health and Health Disparities (5G12MD007603-30)

  • David Jeruzalmi

National Institute of Allergy and Infectious Diseases (Intramural Funding from the Vaccine Research Center)

  • Peter D Kwong

Agency for Science, Technology and Research

  • Yong Zi Tan

National Institutes of Health (R01-MH1148175)

  • Lawrence Shapiro

The authors declare that the funders played no role in this work, including the experimental design, data collection, or data analysis.

Reviewing Editor

  1. Axel T Brunger, Stanford University Medical Center, United States

Version history

  1. Received: December 12, 2017
  2. Accepted: May 17, 2018
  3. Accepted Manuscript published: May 29, 2018 (version 1)
  4. Version of Record published: June 13, 2018 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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  1. Alex J Noble
  2. Venkata P Dandey
  3. Hui Wei
  4. Julia Brasch
  5. Jillian Chase
  6. Priyamvada Acharya
  7. Yong Zi Tan
  8. Zhening Zhang
  9. Laura Y Kim
  10. Giovanna Scapin
  11. Micah Rapp
  12. Edward T Eng
  13. William J Rice
  14. Anchi Cheng
  15. Carl J Negro
  16. Lawrence Shapiro
  17. Peter D Kwong
  18. David Jeruzalmi
  19. Amedee des Georges
  20. Clinton S Potter
  21. Bridget Carragher
(2018)
Routine single particle CryoEM sample and grid characterization by tomography
eLife 7:e34257.
https://doi.org/10.7554/eLife.34257

Share this article

https://doi.org/10.7554/eLife.34257

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