Cargando…
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...
Autores principales: | , , |
---|---|
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 |
_version_ | 1784662812481552384 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8895008 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT peckjaredv highspeedhighresolutiondatacollectionona200kevcryotem AT fayjonathanf highspeedhighresolutiondatacollectionona200kevcryotem AT straussjoshuad highspeedhighresolutiondatacollectionona200kevcryotem |