The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at ∼14 Å resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of ∼100 Å and can be divided into four separate densities. From the side, Wzc is ∼110 Å high and has a distinctive appearance similar to an extracted molar tooth. The upper “crown” region is ∼55 Å high and forms a continuous ring of density. Four unconnected “roots” (∼65 Å high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.

ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.

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, nano-discs, 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 and 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, lipo-polysaccharide biosynthesis and the human major facilitator super-family, 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.

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.