Lipid environment of membrane proteins in cryo-EM based structural analysis

Abstract

Cryoelectron microscopy (cryo-EM) in association with a single particle analysis method (SPA) is now a promising tool to determine the structures of proteins and their macromolecular complexes. The development of direct electron detection cameras and image processing technologies has allowed the structures of many important proteins to be solved at near-atomic resolution or, in some cases, at atomic resolution, by overcoming difficulties in crystallization or low yield of protein production. In the case of membrane-integrated proteins, the proteins were traditionally solubilized and stabilized with various kind of detergents. However, the density of detergent micelles diminished the contrast of membrane proteins in cryo-EM studies and made it difficult to obtain high-resolution structures. To improve the resolution of membrane protein structures in cryo-EM studies, major improvements have been made both in sample preparation techniques and in hardware and software developments. The focus of our review is on improvements which have been made in the various techniques for sample preparation for cryo-EM studies, with a specific interest placed on techniques for mimicking the lipid environment of membrane proteins.

Keywords: Cryoelectron microscopy; Lipid environment; Membrane proteins; Single particle analysis.

Fig. 1

Electron microscopy (EM) for structural biology. a Sample preparation for negative staining EM (left) and cryoelectron microscopy (cryo-EM) (right). In this figure, membrane proteins (green) are associated with membrane lipids (red). b EM images of the hepatitis B small surface antigen: negative staining transmission EM image (left) and cryo-EM image (right). Bar: 100 nm

Fig. 2

Structures of various artificial membranes. Solubilization of membrane proteins with detergents forms micelle structure. Hydrophobic acyl chains interact with the transmembrane surface of membrane proteins. Amphipathic polymer amphipoles are substituted with detergents to form a stable complex in solution. Bicelles are generated by mixing two components. Phospholipids with a long chain form interact with the protein and form a bilayer, and detergents with a short chain fill the rim of the disc. In the nanodisc, two membrane scaffold proteins assemble around detergent-solubilized membrane proteins with lipids to form disc shaped particles. Styrene–maleic acid (SMA) copolymers are polymer-based particles which cover the acyl chains of the lipid bilayer. Membrane proteins assemble into liposomes to form proteoliposomes

Fig. 3

Cryo-EM of the Burkholderia pseudomallei N-type ATPase rotor ring. a–d The two-dimensional class averages the B. pseudomallei c-ring complex, showing a clear correlation between the density of the detergent [n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) 0.88 g/ml; n-dodecyl-β-D-maltopyranoside (DDM) 1.19 g/ml; C12E8 1.04 g/ml] or amphipol (1.3–1.9 g/ml) and the resolution achieved. e, f. Top and side views, respectively, of the rotor ring in LDAO with subunits fitted. Reproduced from Schulz et al. (2017) with permission

Fig. 4

Structure of tripartite double-knot toxin (DkTx)– transient receptor potential cation channel subfamily V member 1 (TRPV1)–lipid complex reconstituted in a nanodisc. In the reconstructed three-dimensional structure (a, left) and its enlarged view (b, right), interactions among tightly bound lipids, amino acid side chains from the TRPV1 and from the DkTx are clearly demonstrated. One DkTx molecule (top, colored purple) interacting with two adjacent TRPV1 subunits (gray) and associated lipids (blue spheres with red and orange colored phosphate head groups). The 2.9-Å resolution of the TRPV1 complex in this study (nanodisc embedded form) was much improved from the 3.8-Å resolution obtained from the amphipol-substituted specimen (Liao et al. 2013). Reproduced from Gao et al. (2016) with permission