Single-particle electron microscopy in the study of membrane protein structure

Abstract

Single-particle electron microscopy (EM) provides the great advantage that protein structure can be studied without the need to grow crystals. However, due to technical limitations, this approach played only a minor role in the study of membrane protein structure. This situation has recently changed dramatically with the introduction of direct electron detection device cameras, which allow images of unprecedented quality to be recorded, also making software algorithms, such as three-dimensional classification and structure refinement, much more powerful. The enhanced potential of single-particle EM was impressively demonstrated by delivering the first long-sought atomic model of a member of the biomedically important transient receptor potential channel family. Structures of several more membrane proteins followed in short order. This review recounts the history of single-particle EM in the study of membrane proteins, describes the technical advances that now allow this approach to generate atomic models of membrane proteins and provides a brief overview of some of the membrane protein structures that have been studied by single-particle EM to date.

Keywords: cryo-electron microscopy; membrane proteins; single-particle electron microscopy; structure determination.

Fig. 1.

Cryo-EM analysis of the conformational dynamics of AMPA receptors in the desensitized state. (a) Class averages of vitrified GluA2 homotetramers in the presence of fluorowillardiine, which stabilizes the receptor in the desensitized state [21]. The averages show that desensitization causes the two dimeric extracellular domains to separate, which then adopt a continuum of conformations. (b) Three 3D maps of GluA2 homotetramers in the desensitized state obtained by 3D classification. ATD, amino-terminal domain; LBD, ligand-binding domain; TMD, transmembrane domain. (b) Adapted from [22] and reprinted by permission from Macmillan Publishers Ltd: Nature, copyright 2014.

Fig. 2.

Early, low-resolution density maps (a–d) and a more recent, intermediate-resolution density map (e) of the IP₃ receptor obtained by single-particle EM as seen from the cytoplasm (top row panels), parallel to the membrane (second row panels) and from the ER lumen (third row panels). The panels in the bottom row show central sections through the density maps at the position indicated by the lines in the panels in the third row, revealing the internal architecture of the receptor. (a) Density map at 24 Å resolution obtained with images of vitrified specimens collected at 120 kV on film, and the 3D reconstruction was calculated with the EMAN (and IMAGIC) software packages [29]. (b) Density map at 30 Å resolution obtained with images of vitrified specimens collected at 100 kV on film, and the 3D reconstruction was calculated with the EMAN software package [31]. (c) Density map at 24 Å resolution obtained with images of vitrified specimens collected at 300 kV on film, and the 3D reconstruction was calculated with the IMAGIC software package [30]. (d) Density map at 30 Å resolution obtained with images of negatively stained specimens collected at 200 kV on film, and the 3D reconstruction was calculated with the IMAGIC software package [27]. (e) Density map at 9.5 Å resolution obtained with images of vitrified specimens collected at 200 kV with a CCD camera, and the 3D reconstruction was calculated with the EMAN software package (EMD 5278) [32]. (a–d) Adapted and reprinted from Serysheva and Ludtke [33], Copyright 2010, with permission from Elsevier.

Fig. 3.

The high quality of data that can be recorded with DDD cameras. (a, b) Average images of rotavirus double-layer particles in vitrified ice obtained from averaging 60 frames of a movie without aligning the frames to each other, showing substantial image blurring (a), and with aligning the frames, largely eliminating image blurring (b). (c and d) Single-particle cryo-EM density maps of transferrin–transferrin receptor complex (gold surface) with docked crystal structure shown as red ribbon diagram (PDB id 1SUV [59]). While the data for the two density maps were collected and processed in essentially the same way and the two density maps have similar resolutions of 7.5 and 7.0 Å, respectively, the map calculated with images recorded on film (c) [60] shows much less structural detail than the map calculated with images recorded with a DDD camera (unpublished data). (a) and (b) Reprinted from Brilot et al. [58], Copyright 2012, with permission from Elsevier.

Fig. 4.

The use of a Fab fragment for structure determination by single-particle cryo-EM. (a and b) Class averages (a) and 10-Å resolution density map (EMD 6087; [85]) (b) of the heterodimeric ABC transporter TmrAB in vitrified ice. (c) Two-dimensional class averages of the TmrAB transporter in complex with Fab AH5, clearly showing additional density for the Fab. (d) In the presence of the Fab, which provides additional mass and a fiducial marker for alignment, the resolution of the density map improved to 8.2 Å (EMD 6085; [85]). (a) and (c) Reprinted from [85] by permission from Macmillan Publishers Ltd: Nature, copyright 2015.

Fig. 5.

Examples of membrane protein structures determined by single-particle cryo-EM. Density maps are shown as gold surfaces and atomic models as red ribbon diagrams. The black lines indicate the approximate position of the membrane. (a) Density map of the 2.2-MDa homotetrameric ryanodine receptor at 4.8 Å resolution (EMD 6107, PDB 3J8E; [91]). (b) Density maps of V-type ATPases obtained from data collected with a CCD camera at 9.7 Å (EMD 5335, PDB 3J0J; [92]) (left panel) and from data collected with a DDD camera at 6.9 Å (EMD 6284, PDB 3J9 T; [93]) (right panel). (c) Density map of respiratory complex I at 4.95 Å resolution (EMD 2676, PDB 4UQ8; [94]). (d) Density map of the 300-kDa homotetrameric TRPV1 channel at 3.4 Å resolution (EMD 5778, PDB 3J5P; [63]).