Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales

David P. Klebl; Molly S. C. Gravett; Dimitrios Kontziampasis; David J. Wright; Robin S. Bon; Diana C. F. Monteiro; Martin Trebbin; Frank Sobott; Howard D. White; Michele C. Darrow; Rebecca F. Thompson; Stephen P. Muench

doi:10.1016/j.str.2020.07.018

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales

David P. Klebl, Molly S. C. Gravett, Dimitrios Kontziampasis, David J. Wright, Robin S. Bon, Diana C. F. Monteiro, Martin Trebbin, Frank Sobott, Howard D. White, Michele C. Darrow, Rebecca F. Thompson (Lead / Corresponding author), Stephen P. Muench (Lead / Corresponding author)

Mechanical and Industrial Engineering

Research output: Contribution to journal › Article › peer-review

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Abstract

A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

Original language	English
Pages (from-to)	1238-1248.e4
Number of pages	16
Journal	Structure
Volume	28
Issue number	11
Early online date	18 Aug 2020
DOIs	https://doi.org/10.1016/j.str.2020.07.018
Publication status	Published - 3 Nov 2020

Keywords

air-water-interface
cryoEM
HSPD1
ribosome
sample preparation

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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10.1016/j.str.2020.07.018Licence: CC BY

Final Published VersionFinal published version, 3.26 MBLicence: CC BY

Cite this

@article{2f3d765ef0144eeab19bf650add09edb,

title = "Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales",

abstract = "A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.",

keywords = "air-water-interface, cryoEM, HSPD1, ribosome, sample preparation",

author = "Klebl, {David P.} and Gravett, {Molly S. C.} and Dimitrios Kontziampasis and Wright, {David J.} and Bon, {Robin S.} and Monteiro, {Diana C. F.} and Martin Trebbin and Frank Sobott and White, {Howard D.} and Darrow, {Michele C.} and Thompson, {Rebecca F.} and Muench, {Stephen P.}",

note = "Funding Information: We thank Dr. Hao Shao and Dr. Jason Gestwicki (UCSF) for providing the HSPD1 plasmid used in this work. D.P.K. and M.S.C.G. are PhD students on the Wellcome Trust 4-year PhD program in The Astbury Center funded by The University of Leeds . D.J.W. and D.K. were funded by the BBSRC ( BB/P020208/1 and BB/P026397/1 , respectively). The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust ( 108466/Z/15/Z ). Maps have been deposited within the EMDB; ribosome 30S at 13 ms, 54 ms, 200 ms, and blotted (EMDB: 10871, 10872, 10873, and 10874, respectively), ribosome 50S at 13 ms, 54 ms, 200 ms, and blotted (EMDB: 10875, 10876, 10877, and 10878, respectively), ribosome 70S at 13 ms, 54 ms, 200 ms, and blotted (EMDB: 10879, 10880, 10881, and 10882), and HSPD1 at 6 ms, 50 ms, 54 ms, and blotted (EMDB: 10883, 10884, 10885, and 10886). Copyright: {\textcopyright} 2020 The Authors. Published by Elsevier Ltd.",

year = "2020",

month = nov,

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doi = "10.1016/j.str.2020.07.018",

language = "English",

volume = "28",

pages = "1238--1248.e4",

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number = "11",

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AU - Klebl, David P.

AU - Gravett, Molly S. C.

AU - Kontziampasis, Dimitrios

AU - Wright, David J.

AU - Bon, Robin S.

AU - Monteiro, Diana C. F.

AU - Trebbin, Martin

AU - Sobott, Frank

AU - White, Howard D.

AU - Darrow, Michele C.

AU - Thompson, Rebecca F.

AU - Muench, Stephen P.

N1 - Funding Information: We thank Dr. Hao Shao and Dr. Jason Gestwicki (UCSF) for providing the HSPD1 plasmid used in this work. D.P.K. and M.S.C.G. are PhD students on the Wellcome Trust 4-year PhD program in The Astbury Center funded by The University of Leeds . D.J.W. and D.K. were funded by the BBSRC ( BB/P020208/1 and BB/P026397/1 , respectively). The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust ( 108466/Z/15/Z ). Maps have been deposited within the EMDB; ribosome 30S at 13 ms, 54 ms, 200 ms, and blotted (EMDB: 10871, 10872, 10873, and 10874, respectively), ribosome 50S at 13 ms, 54 ms, 200 ms, and blotted (EMDB: 10875, 10876, 10877, and 10878, respectively), ribosome 70S at 13 ms, 54 ms, 200 ms, and blotted (EMDB: 10879, 10880, 10881, and 10882), and HSPD1 at 6 ms, 50 ms, 54 ms, and blotted (EMDB: 10883, 10884, 10885, and 10886). Copyright: © 2020 The Authors. Published by Elsevier Ltd.

PY - 2020/11/3

Y1 - 2020/11/3

N2 - A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

AB - A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

KW - air-water-interface

KW - cryoEM

KW - HSPD1

KW - ribosome

KW - sample preparation

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U2 - 10.1016/j.str.2020.07.018

DO - 10.1016/j.str.2020.07.018

M3 - Article

C2 - 32814033

AN - SCOPUS:85090243631

SN - 0969-2126

VL - 28

SP - 1238-1248.e4

JO - Structure

JF - Structure

IS - 11

ER -

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales

Abstract

Keywords

ASJC Scopus subject areas

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Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales

Cite this