Coming Clean and Avoiding Bubble Trouble–Using Detergents Wisely in the Purification of Membrane Proteins for Cryo-EM Studies

Abstract

Detergent solubilisation remains the most commonly used but potentially problematic method to extract membrane proteins from lipid bilayers for Cryo-EM studies. Although recent advances have introduced excellent alternatives—such as amphipols, nanodiscs and SMALPs—the use of detergents is often necessary for intermediate steps. In this paper, we share our experiences working with detergent-solubilised samples within the modern Cryo-EM structural pipeline from the perspective of an EM specialist. Our aim is to inform novice users about potential challenges they may encounter. Drawing on specific examples from a variety of biological membrane systems, including Magnesium channels, lipopolysaccharide biosynthesis, and the human major facilitator superfamily transporters, we describe how the intrinsic properties of detergent-extracted samples can affect protein purification, Cryo-EM grid preparation (including the formation of vitreous ice) and the reconstitution of proteins into micelles. We also discuss how these unique characteristics can impact different stages of structural analysis and lead to complications in single-particle averaging software analysis. For each case, we present our insights into the underlying causes and suggest possible mitigations or alternative approaches.

Publication DOI: https://doi.org/10.3390/biom15091315
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences
Aston University (General)
Additional Information: Copyright © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Publication ISSN: 2218-273X
Data Access Statement: The authors will deposit the Wzz C6 MRC volume and modelling PDB to the EMDB on acceptance. Raw EM data particle stacks will be archived in line with journal policy.
Last Modified: 06 Oct 2025 07:30
Date Deposited: 26 Sep 2025 08:47
Full Text Link:
Related URLs: https://www.mdp ... -273X/15/9/1315 (Publisher URL)
PURE Output Type: Article
Published Date: 2025-09-12
Published Online Date: 2025-09-12
Accepted Date: 2025-09-10
Authors: Chen, Bowen
Harrison, Peter
Kargas, Vasileios
Pollock, Naomi (ORCID Profile 0000-0003-0345-0923)
Ford, Robert C.
Prince, Stephen M.
Collins, Richard F.

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