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High-speed high-resolution data collection on a 200 keV cryo-TEM

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, meth...

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Autores principales: Peck, Jared V., Fay, Jonathan F., Strauss, Joshua D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8895008/
https://www.ncbi.nlm.nih.gov/pubmed/35371504
http://dx.doi.org/10.1107/S2052252522000069
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author Peck, Jared V.
Fay, Jonathan F.
Strauss, Joshua D.
author_facet Peck, Jared V.
Fay, Jonathan F.
Strauss, Joshua D.
author_sort Peck, Jared V.
collection PubMed
description 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 (∼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 Å 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 Å 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.
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spelling pubmed-88950082022-03-31 High-speed high-resolution data collection on a 200 keV cryo-TEM Peck, Jared V. Fay, Jonathan F. Strauss, Joshua D. IUCrJ Research Papers 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 (∼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 Å 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 Å 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. International Union of Crystallography 2022-01-29 /pmc/articles/PMC8895008/ /pubmed/35371504 http://dx.doi.org/10.1107/S2052252522000069 Text en © Peck, Fay and Strauss 2022 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
spellingShingle Research Papers
Peck, Jared V.
Fay, Jonathan F.
Strauss, Joshua D.
High-speed high-resolution data collection on a 200 keV cryo-TEM
title High-speed high-resolution data collection on a 200 keV cryo-TEM
title_full High-speed high-resolution data collection on a 200 keV cryo-TEM
title_fullStr High-speed high-resolution data collection on a 200 keV cryo-TEM
title_full_unstemmed High-speed high-resolution data collection on a 200 keV cryo-TEM
title_short High-speed high-resolution data collection on a 200 keV cryo-TEM
title_sort high-speed high-resolution data collection on a 200 kev cryo-tem
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8895008/
https://www.ncbi.nlm.nih.gov/pubmed/35371504
http://dx.doi.org/10.1107/S2052252522000069
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