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Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids
Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the s...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier Inc.
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400742/ https://www.ncbi.nlm.nih.gov/pubmed/28254381 http://dx.doi.org/10.1016/j.jsb.2017.02.008 |
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author | Snijder, Joost Borst, Andrew J. Dosey, Annie Walls, Alexandra C. Burrell, Anika Reddy, Vijay S. Kollman, Justin M. Veesler, David |
author_facet | Snijder, Joost Borst, Andrew J. Dosey, Annie Walls, Alexandra C. Burrell, Anika Reddy, Vijay S. Kollman, Justin M. Veesler, David |
author_sort | Snijder, Joost |
collection | PubMed |
description | Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM. |
format | Online Article Text |
id | pubmed-5400742 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Elsevier Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-54007422018-04-01 Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids Snijder, Joost Borst, Andrew J. Dosey, Annie Walls, Alexandra C. Burrell, Anika Reddy, Vijay S. Kollman, Justin M. Veesler, David J Struct Biol Article Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM. Elsevier Inc. 2017-04 2017-02-22 /pmc/articles/PMC5400742/ /pubmed/28254381 http://dx.doi.org/10.1016/j.jsb.2017.02.008 Text en © 2017 Elsevier Inc. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Snijder, Joost Borst, Andrew J. Dosey, Annie Walls, Alexandra C. Burrell, Anika Reddy, Vijay S. Kollman, Justin M. Veesler, David Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
title | Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
title_full | Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
title_fullStr | Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
title_full_unstemmed | Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
title_short | Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
title_sort | vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400742/ https://www.ncbi.nlm.nih.gov/pubmed/28254381 http://dx.doi.org/10.1016/j.jsb.2017.02.008 |
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