Coming clean and avoiding bubble trouble - using detergents wisely in the purification of membrane proteins for Cryo-EM studies

DOI: 10.3390/biom15091315

Authors: Bowen Chen (The University of Manchester) , Peter Harrison (Diamond Light Source) , Vasileios Kargas (University of Cambridge) , Naomi Pollock (Aston University) , Robert C. Ford (The University of Manchester) , Stephen M. Prince (The University of Manchester) , Richard F. Collins (The University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biomolecules , VOL 15

State: Published (Approved)
Published: September 2025
Diamond Proposal Number(s): 29255 , 36408 , 27436 , 33941

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.

Journal Keywords: cryo electron microscopy; Cryo-EM grid preparation; membrane proteins; detergent purification; membrane protein structure; Wzz lipopolysaccharide biosynthesis; CorA Magnesium channel structure; transmembrane helices; oligomeric complexes; single particle

Subject Areas: Biology and Bio-materials, Chemistry

Diamond Offline Facilities: Membrane Protein Laboratory (MPL), Electron Bio-Imaging Centre (eBIC)
Instruments: Krios I-Titan Krios I at Diamond , Krios II-Titan Krios II at Diamond , Krios III-Titan Krios III at Diamond , Krios IV-Titan Krios IV at Diamond , Krios V-Titan Krios V at Diamond

Added On: 17/09/2025 18:51

Documents:
biomolecules-15-01315-v3.pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Microscopy Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)