Single-particle reconstruction of biological macromolecules in electron microscopy--30 years

Abstract

This essay gives the autho's personal account on the development of concepts underlying single-particle reconstruction, a technique in electron microscopy of macromolecular assemblies with a remarkable record of achievements as of late. The ribosome proved to be an ideal testing ground for the development of specimen preparation methods, cryo-EM techniques, and algorithms, with discoveries along the way as a rich reward. Increasingly, cryo-EM and single-particle reconstruction, in combination with classification techniques, is revealing dynamic information on functional molecular machines uninhibited by molecular contacts.

Fig. 1

Cross-correlation function, showing a peak indicating the relative position of two successive images taken of the same carbon film. Data reproduced from Frank (1970).

Fig. 2

Averaging of 40S subunits from HeLa cell ribosomes. (a) Electron micrograph of negatively stained subunits, with “L” denoting left-facing views selected for alignment and averaging. (b, c) Averages of halfsets of 40 particles each; (d) variance map; (e) total average; (f) three measures of resolution: R, R factor; Δ, Euclidean distance; δϕ, DPR indicating a resolution of 20 Å at 45°. Data reproduced from Frank et al. (1981b); panels R and Δ in (f) are from unpublished data.

Fig. 3

Example of Fourier shell correlation curves, with resolution criterion (FSC=0·5) indicated. Data reproduced from Sengupta et al. (2008).

Fig. 4

Factor map (factors 1 versus 2) resulting from correspondence analysis of L. polyphemus hemocyanin half molecules. Averages of images falling into demarcated clusters are shown in the four corners (a–d). (a) and (c) are interpreted as two rocking positions of molecules lying on one (“flip”) side, while (b) and (d) relate to rocking positions on the other (“flop”) side. Adapted from Frank (1984).

Fig. 5

Random–conical reconstruction. (a) Principle of the random–conical data collection method. Two images are taken of the same field of molecules. Only molecules are considered that present the same view on the grid. Azimuthal angles are obtained by aligning the images of the untilted micrograph. Thus, with both azimuth and tilt angles known, the Fourier transform of each projection can be properly placed into the 3D Fourier reference frame of the molecule. From J. Frank (unpublished hand-drawing on overhead transparency, 1979). (b–d) Density map of the 50S ribosomal subunit from E. coli, the first 3D reconstruction using the random–conical data collection method. (a) Surface representation of intersubunit face; (b, c) higher-threshold solid model obtained by stacking of contoured slices, viewed from front and back. The subunit was negatively stained with uranyl acetate and air-dried, which accounts for the partial flattening. The ridge of the deep groove running horizontally, termed interface canyon, is created by the helix 69 of 23S rRNA, as later recognized when the X-ray structure of the large subunit was solved. Annotations refer to morphological details; for example, pocket ‘P2’ was suggested to be the peptidyl transferase center and CP the central protuberance. Data reproduced from Radermacher et al. (1987).

Fig. 6

Cryo-EM reconstruction of the (empty) E. coli ribosome from 4300 particle images, at 25-Å resolution. Cut-open density maps depict the way mRNA, tRNAs, and the polypeptide chain were thought to be positioned during protein synthesis, a model that has stood up to the test of time. Landmarks on the large subunit (50S): CP, central protuberance; St, stalk base; L1, L1 stalk; IC, interface canyon; T, polypeptide exit tunnel; T1, T2, two putative branches of the tunnel exit; E1, E2, corresponding exit sites. Landmarks on the small subunit (30S): h, head; p, platform; ch, mRNA channel. A, P, putative positions of A- and P-site tRNA. Data reproduced from Frank et al. (1995).

Fig. 7

Classification of ribosome complexes with or without EF-G by two methods: supervised and unsupervised classification. The experimental data set contains two states of the ribosome, distinguished as follows: (one) contains no EF-G; the A and P sites are occupied by tRNAs in the classic state; and the ribosome is in macrostate I (i.e., unratcheted), the other has EF-G bound; there is a single tRNA in P/E position; and the ribosome is in macrostate II (i.e., ratcheted). (a) Results of classification with the maximum likelihood (ML) method, with four classes specified. (b) Cross-reference between classes from ML and those derived from dividing the correlation histogram obtained by supervised classification. (c) Results of supervised classification using two reference maps, in ribosome macrostates I and II but without EF-G. Data reproduced from Scheres et al. (2007).

Fig. 8

Homology modeling of the proteins and RNA of the 80S eukaryotic ribosome from the fungus T. lanuginosus, based on the sequence of a close relative, Saccharomyces cerevisiae, and on the X-ray structure of E. coli (Taylor et al. 2009). Structure in red seen in front is eEF2.

Fig. 9

Cryo-EM reconstruction of the 70S ribosome of E. coli, with Phe-tRNA^Phe·EF-Tu·GDP bound in the presence of kirromycin (LeBarron et al. 2008). Yellow: 30S subunit; blue: 50S subunit. Highlighted peripheral ribosomal protein S2 (encircled) is compared with its X-ray structure (orange) presented in the same orientation. Arrow points to an outermost helix that is missing in the density map, presumably because of disorder, while the rest of the structure is intact.