High-speed high-resolution data collection on a 200 keV cryo-TEM
Open Access
- 29 January 2022
- journal article
- research article
- Published by International Union of Crystallography (IUCr) in IUCrJ
- Vol. 9 (2), 243-252
- https://doi.org/10.1107/s2052252522000069
Abstract
Limitations to successful single-particle cryo-electron microscopy (cryo-EM) projects include stable sample generation, production of quality cryo-EM grids with randomly oriented particles embedded in thin vitreous ice and access to microscope time. To address the limitation of microscope time, methodologies to more efficiently collect data on a 200 keV Talos Arctica cryo-transmission electron microscope at speeds as fast as 720 movies per hour (similar to 17 000 per day) were tested. In this study, key parameters were explored to increase data collection speed including: (1) using the beam-image shift method to acquire multiple images per stage position, (2) employing UltrAufoil TEM grids with R0.6/1 hole spacing, (3) collecting hardware-binned data and (4) adjusting the image shift delay factor in SerialEM. Here, eight EM maps of mouse apoferritin at 1.8-1.9 angstrom resolution were obtained in the analysis with data collection times for each dataset ranging from 56 min to 2 h. An EM map of mouse apoferritin at 1.78 angstrom was obtained from an overnight data collection at a speed of 500 movies per hour and subgroup analysis performed, with no significant variation observed in data quality by image shift distance and image shift delay. The findings and operating procedures detailed herein allow for rapid turnover of single-particle cryo-EM structure determination.Keywords
Funding Information
- National Cancer Institute of the National Institutes of Health (P30CA016086)
- Core Facility Advocacy Committee and Office of Research Technologies, UNC Chapel Hill School of Medicine
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