eLife (May 2018)
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
Affiliations
- Alex J Noble
- ORCiD
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Venkata P Dandey
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Hui Wei
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Julia Brasch
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
- Jillian Chase
- Department of Chemistry and Biochemistry, City College of New York, New York, United States; Program in Biochemistry, The Graduate Center of the City University of New York, New York, United States
- Priyamvada Acharya
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Maryland, United States
- Yong Zi Tan
- ORCiD
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
- Zhening Zhang
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Laura Y Kim
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Giovanna Scapin
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Structural Chemistry and Chemical Biotechnology, Merck & Co., Inc, New Jersey, United States
- Micah Rapp
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
- Edward T Eng
- ORCiD
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- William J Rice
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Anchi Cheng
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Carl J Negro
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
- Lawrence Shapiro
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
- Peter D Kwong
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Maryland, United States
- David Jeruzalmi
- ORCiD
- Department of Chemistry and Biochemistry, City College of New York, New York, United States; Program in Biochemistry, The Graduate Center of the City University of New York, New York, United States; Program in Biology, The Graduate Center of the City University of New York, New York, United States; Program in Chemistry, The Graduate Center of the City University of New York, New York, United States
- Amedee des Georges
- Department of Chemistry and Biochemistry, City College of New York, New York, United States; Program in Biochemistry, The Graduate Center of the City University of New York, New York, United States; Program in Chemistry, The Graduate Center of the City University of New York, New York, United States; Advanced Science Research Center, The Graduate Center of the City University of New York, New York, United States
- Clinton S Potter
- ORCiD
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
- Bridget Carragher
- ORCiD
- National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, United States
- DOI
- https://doi.org/10.7554/eLife.34257
- Journal volume & issue
-
Vol. 7
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
