Cryo-EM analysis of a membrane protein embedded in the liposome

Abstract

Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.

Keywords: cryo-EM; graphene grids; membrane protein; proteoliposome; structural biology.

Fig. 1.

Optimization of proteoliposome cryo-sample preparation. (A) A schematic illustration of size selection of proteoliposomes using SEC. When the mixture of proteoliposomes and detergent micelles pass through different resins for SEC in the absence of supplemented detergents, liposomes are separated based on their diameters and detergents are removed. Please refer to SI Appendix, Fig. S1 (SI Appendix) for more trials of proteoliposome optimization. (B) Representative cryo-EM micrographs and size distribution of AcrB proteoliposomes prepared using manually packed Sephadex G-50 (Left) and G-100 columns (Right). Histograms are calculated from five holes of each sample. (C–E) Micrographs of AcrB proteoliposomes loaded on commercial Qauntifoil grids with (C) single- and (D) multiapplication approaches, and (E) on a graphene with single-application. The cartoon illustration for proteoliposome distribution in the holes by various sample preparation methods are presented below the corresponding panel.

Fig. 2.

Deep 2D classification for selection of good protein particles embedded in liposomes. Protein particles (indicated by red squares) were extracted from 5,757 motion-corrected micrographs. (Scale bar: 50 nm.) After a gross 2D classification for all extracted particles, a “deep” 2D classification strategy was applied to the 2D classes containing over 1,000 particles. Particles in deep 2D classes with clear protein signal were collected (red boxes). Final 2D averages from the selected particles display clear secondary structure features. (Scale bar: 10 nm.) More details are provided in SI Appendix, Fig. S3.

Fig. 3.

Single-particle reconstruction of AcrB embedded in proteoliposomes. (A) Top view and side view of the single-particle reconstruction of AcrB at 3.9 Å resolution. (B) EM map with low display threshold reveals lipid bilayer signal. The AcrB structure (PDB code: 1IWG) is docked into the density (before B-factor sharpening) contoured at 1.0σ. (C) Local resolution map of the AcrB reconstruction. The unit for resolutions shown on the right is Å. (D) Representative densities of two alpha helices and two beta strands of AcrB, shown as blue mesh with indicated contour level.

Fig. 4.

AcrB surrounded by different membrane curvatures exhibits identical structure. (A) 2D class averages shown different membrane curvatures around AcrB. Two representative classes stand out. (B) Reconstruction of the membrane fraction. The 3D reconstructions of the particles in the two 2D classes shown in A are low-pass filtered to 7 Å. Membrane curvatures are indicated by red and black, dashed line. (C) AcrB from the two classes are nearly identical. The two 3D reconstructions are overlaid, with the respective membrane curvatures indicated by red and black dashed lines.