Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines

Aurélien Deniaud; Burak V Kabasakal; Joshua C Bufton; Christiane Schaffitzel

doi:10.1007/978-3-031-52193-5_12

Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines

Aurélien Deniaud^*
, Burak V Kabasakal
, Joshua C Bufton
, Christiane Schaffitzel^*

^*Corresponding author for this work

School of Biochemistry

Research output: Chapter in Book/Report/Conference proceeding › Chapter in a book

1 Citation (Scopus)

33 Downloads (Pure)

Abstract

High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.

Original language	English
Title of host publication	Advanced Technologies for Protein Complex Production and Characterization
Editors	M. Cristina Vega, Francisco J. Fernandez
Publisher	Springer, Cham
Pages	173-190
Number of pages	18
Volume	2
ISBN (Electronic)	9783031521935
ISBN (Print)	9783031521928
DOIs	https://doi.org/10.1007/978-3-031-52193-5_12
Publication status	E-pub ahead of print - 21 Mar 2024

Publication series

Name	Advances in Experimental Medicine and Biology
Volume	3234
ISSN (Print)	0065-2598
ISSN (Electronic)	2214-8019

Bibliographical note

Publisher Copyright:
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024.

Research Groups and Themes

Bristol BioDesign Institute

Keywords

Cryoelectron Microscopy/methods
Electrons
Membrane Proteins
Freezing
Specimen Handling/methods
Macromolecular Substances

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10.1007/978-3-031-52193-5_12Licence: Unspecified

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This is the accepted author manuscript (AAM). The final published version (Version of Record) can be found on the publisher's website. The copyright of any third-party content, such as images, remains with the copyright holder.
Accepted author manuscript, 997 KBLicence: Unspecified

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Membrane protein insertion and quality control by the bacterial holo-translocon and FtsH chaperone/protease complex
Berger-Schaffitzel, C. H. (Principal Investigator)
9/01/17 → 8/07/20
Project: Research

Cite this

Deniaud, A., Kabasakal, B. V., Bufton, J. C., & Schaffitzel, C. (2024). Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines. In M. C. Vega, & F. J. Fernandez (Eds.), Advanced Technologies for Protein Complex Production and Characterization (Vol. 2, pp. 173-190). (Advances in Experimental Medicine and Biology; Vol. 3234). Springer, Cham. Advance online publication. https://doi.org/10.1007/978-3-031-52193-5_12

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abstract = "High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.",

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author = "Aur{\'e}lien Deniaud and Kabasakal, \{Burak V\} and Bufton, \{Joshua C\} and Christiane Schaffitzel",

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Deniaud, A, Kabasakal, BV, Bufton, JC & Schaffitzel, C 2024, Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines. in MC Vega & FJ Fernandez (eds), Advanced Technologies for Protein Complex Production and Characterization . vol. 2, Advances in Experimental Medicine and Biology, vol. 3234, Springer, Cham, pp. 173-190. https://doi.org/10.1007/978-3-031-52193-5_12

Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines. / Deniaud, Aurélien; Kabasakal, Burak V; Bufton, Joshua C et al.
Advanced Technologies for Protein Complex Production and Characterization . ed. / M. Cristina Vega; Francisco J. Fernandez. Vol. 2 Springer, Cham, 2024. p. 173-190 (Advances in Experimental Medicine and Biology; Vol. 3234).

Research output: Chapter in Book/Report/Conference proceeding › Chapter in a book

TY - CHAP

T1 - Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines

AU - Deniaud, Aurélien

AU - Kabasakal, Burak V

AU - Bufton, Joshua C

AU - Schaffitzel, Christiane

PY - 2024/3/21

Y1 - 2024/3/21

N2 - High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.

AB - High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.

KW - Cryoelectron Microscopy/methods

KW - Electrons

KW - Membrane Proteins

KW - Freezing

KW - Specimen Handling/methods

KW - Macromolecular Substances

U2 - 10.1007/978-3-031-52193-5_12

DO - 10.1007/978-3-031-52193-5_12

M3 - Chapter in a book

C2 - 38507207

SN - 9783031521928

VL - 2

T3 - Advances in Experimental Medicine and Biology

SP - 173

EP - 190

BT - Advanced Technologies for Protein Complex Production and Characterization

A2 - Vega, M. Cristina

A2 - Fernandez, Francisco J.

PB - Springer, Cham

ER -

Deniaud A, Kabasakal BV, Bufton JC, Schaffitzel C. Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines. In Vega MC, Fernandez FJ, editors, Advanced Technologies for Protein Complex Production and Characterization . Vol. 2. Springer, Cham. 2024. p. 173-190. (Advances in Experimental Medicine and Biology). Epub 2024 Mar 21. doi: 10.1007/978-3-031-52193-5_12

Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines

Abstract

Publication series

Bibliographical note

Research Groups and Themes

Keywords

Access to Document

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Projects

Membrane protein insertion and quality control by the bacterial holo-translocon and FtsH chaperone/protease complex

Cite this