A cryo-EM grid preparation device for time-resolved structural studies

Dimitrios Kontziampasis; David P. Klebl; Matthew G. Iadanza; Charlotte A. Scarff; Florian Kopf; Frank Sobott; Diana C. F. Monteiro; Martin Trebbin; Stephen P. Muench; Howard D. White

doi:10.1101/563254

A cryo-EM grid preparation device for time-resolved structural studies

Dimitrios Kontziampasis
, David P. Klebl
, Matthew G. Iadanza
, Charlotte A. Scarff
, Florian Kopf
, Frank Sobott
, Diana C. F. Monteiro
, Martin Trebbin
, Stephen P. Muench (Lead / Corresponding author)
, Howard D. White (Lead / Corresponding author)

Research output: Working paper/Preprint › Preprint

Abstract

Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 Å reconstruction. Rapid mixing can be achieved by blot and spray or mix and spray approaches with a delay of ~10 ms, providing greater temporal resolution than previously reported approaches.

Original language	English
Publisher	BioRxiv
Pages	1-22
Number of pages	22
DOIs	https://doi.org/10.1101/563254
Publication status	Published - 4 Mar 2019

Keywords

microscope hardware
sample preparation
structural biology
time-resolved cryo-EM
voltage-assisted spraying

ASJC Scopus subject areas

General Chemistry
Biochemistry
General Materials Science
Condensed Matter Physics

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10.1101/563254Licence: CC BY

Cite this

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title = "A cryo-EM grid preparation device for time-resolved structural studies",

abstract = "Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 {\AA} reconstruction. Rapid mixing can be achieved by blot and spray or mix and spray approaches with a delay of \textasciitilde{}10 ms, providing greater temporal resolution than previously reported approaches.",

keywords = "microscope hardware, sample preparation, structural biology, time-resolved cryo-EM, voltage-assisted spraying",

author = "Dimitrios Kontziampasis and Klebl, \{David P.\} and Iadanza, \{Matthew G.\} and Scarff, \{Charlotte A.\} and Florian Kopf and Frank Sobott and Monteiro, \{Diana C. F.\} and Martin Trebbin and Muench, \{Stephen P.\} and White, \{Howard D.\}",

note = "Copyright: The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.",

year = "2019",

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doi = "10.1101/563254",

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AU - Kontziampasis, Dimitrios

AU - Klebl, David P.

AU - Iadanza, Matthew G.

AU - Scarff, Charlotte A.

AU - Kopf, Florian

AU - Sobott, Frank

AU - Monteiro, Diana C. F.

AU - Trebbin, Martin

AU - Muench, Stephen P.

AU - White, Howard D.

N1 - Copyright: The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

PY - 2019/3/4

Y1 - 2019/3/4

N2 - Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 Å reconstruction. Rapid mixing can be achieved by blot and spray or mix and spray approaches with a delay of ~10 ms, providing greater temporal resolution than previously reported approaches.

AB - Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 Å reconstruction. Rapid mixing can be achieved by blot and spray or mix and spray approaches with a delay of ~10 ms, providing greater temporal resolution than previously reported approaches.

KW - microscope hardware

KW - sample preparation

KW - structural biology

KW - time-resolved cryo-EM

KW - voltage-assisted spraying

UR - https://www.scopus.com/pages/publications/85074613976

U2 - 10.1101/563254

DO - 10.1101/563254

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EP - 22

BT - A cryo-EM grid preparation device for time-resolved structural studies

PB - BioRxiv

ER -

A cryo-EM grid preparation device for time-resolved structural studies

Abstract

Keywords

ASJC Scopus subject areas

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A cryo-EM grid preparation device for time-resolved structural studies

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