Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Snijder, Joost, Borst, Andrew J., Dosey, Annie, Walls, Alexandra C., Burrell, Anika, Reddy, Vijay S., Kollman, Justin M., Veesler, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier Inc. 2017
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
_version_ 1783230898391482368
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
work_keys_str_mv AT snijderjoost vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT borstandrewj vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT doseyannie vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT wallsalexandrac vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT burrellanika vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT reddyvijays vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT kollmanjustinm vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids
AT veeslerdavid vitrificationaftermultipleroundsofsampleapplicationandblottingimprovesparticledensityoncryoelectronmicroscopygrids