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Time-resolved cryoEM using Spotiton
Venkata P. Dandey, View ORCID ProfileWilliam C. Budell, Hui Wei, Daija Bobe, Kashyap Maruthi, Mykhailo Kopylov, View ORCID ProfileEdward Eng, Peter A. Kahn, Jenny E. Hinshaw, Nidhi Kundu, Crina M. Nimigean, Chen Fan, Nattakan Sukomon, Seth Darst, Ruth Saecker, James Chen, Brandon Malone, Clinton S. Potter, Bridget Carragher
doi: https://doi.org/10.1101/2020.04.20.050518
Venkata P. Dandey
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
William C. Budell
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
Hui Wei
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
Daija Bobe
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
Kashyap Maruthi
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
Mykhailo Kopylov
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
Edward Eng
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
Peter A. Kahn
2Engineering Arts LLC, Arizona, USA
Jenny E. Hinshaw
3Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 20892, USA
Nidhi Kundu
3Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 20892, USA
Crina M. Nimigean
4Department of Anesthesiology, Weill Cornell Medical College, New York, NY, 10065 USA
Chen Fan
4Department of Anesthesiology, Weill Cornell Medical College, New York, NY, 10065 USA
Nattakan Sukomon
4Department of Anesthesiology, Weill Cornell Medical College, New York, NY, 10065 USA
Seth Darst
5Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA
Ruth Saecker
5Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA
James Chen
5Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA
Brandon Malone
5Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA
Clinton S. Potter
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
6Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
Bridget Carragher
1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave, New York, NY, 10027, USA
6Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
Abstract
We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We demonstrate the utility of this approach for four biological systems where short-lived states are of high interest.
Competing Interest Statement
B.C./C.S.P. have a commercial relationship with STPL, a company that produces a commercially available instrument, Chameleon, that is based on the Spotiton prototype.
Copyright
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Posted April 21, 2020.
Time-resolved cryoEM using Spotiton
Venkata P. Dandey, William C. Budell, Hui Wei, Daija Bobe, Kashyap Maruthi, Mykhailo Kopylov, Edward Eng, Peter A. Kahn, Jenny E. Hinshaw, Nidhi Kundu, Crina M. Nimigean, Chen Fan, Nattakan Sukomon, Seth Darst, Ruth Saecker, James Chen, Brandon Malone, Clinton S. Potter, Bridget Carragher
bioRxiv 2020.04.20.050518; doi: https://doi.org/10.1101/2020.04.20.050518
Time-resolved cryoEM using Spotiton
Venkata P. Dandey, William C. Budell, Hui Wei, Daija Bobe, Kashyap Maruthi, Mykhailo Kopylov, Edward Eng, Peter A. Kahn, Jenny E. Hinshaw, Nidhi Kundu, Crina M. Nimigean, Chen Fan, Nattakan Sukomon, Seth Darst, Ruth Saecker, James Chen, Brandon Malone, Clinton S. Potter, Bridget Carragher
bioRxiv 2020.04.20.050518; doi: https://doi.org/10.1101/2020.04.20.050518
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