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.