Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction

Abstract

Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as "single-particle reconstruction." During the past two decades, this technique has become an important tool for 3D structure determination, but it generally has not been possible to determine atomic models. In principle, individual molecular images contain high-resolution information contaminated by a much higher level of noise. In practice, it has been unclear whether current averaging methods are adequate to extract this information from the background. We present here a reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle ("double-layer particle" or DLP) at a resolution suitable for interpretation by an atomic model. The result establishes single-particle reconstruction as a high-resolution technique. We show by direct comparison that the cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP is similar in clarity to a 3.8-A resolution map obtained from x-ray crystallography. At this resolution, most of the amino acid side chains produce recognizable density. The icosahedral symmetry of the particle was an important factor in achieving this resolution in the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell biology.

Fig. 1.

Image of rotavirus DLPs frozen in ice over a hole in C-flat carbon grids. The arrows indicate particles that differ significantly in appearance from other particles in the image, indicating partially damaged particles. These particles were not used for further processing. More subtle damage to particles that is not readily visible may be one reason for the lower correlation coefficients with the reference seen for many particles (Fig. 5).

Fig. 2.

VP6 trimers in the DLP. (a) Rotavirus DLP structure filtered at 20 Å. VP6 trimers involved in the 13-fold nonicosahedral averaging are indicated by letters. The T trimer coincides with one of the icosahedral threefold axes and contains only one unique VP6 monomer. The other four trimers contain the additional 12 VP6 molecules that were used for nonicosahedral averaging. (b) T trimer of VP6, filtered at 7-Å resolution, with a bundle of three generic helices docked into easily identifiable density features; 12 other bundles were docked in equivalent density in the other four VP6 trimers. (c) Overview of the 13-fold averaged VP6 trimer at 3.8-Å resolution. Different areas are highlighted and labeled with numbers. They are shown in more detail in d and e and in Fig. 6. (d) Density in area 1 before 13-fold averaging, shown with the atomic model of VP6, determined by using the x-ray density map (B. McLain, E.S., R.B., and S.C.H., unpublished data). (e) Same as in d but after 13-fold averaging. All structures were prepared with UCSF CHIMERA (35).

Fig. 3.

Density map of part of the VP2 portion of DLP. The map was filtered at 4.5-Å resolution and sharpened by using a B factor of −450 Ų. The quality of the map is similar to that of VP6 before nonicosahedral averaging (Fig. 2d).

Fig. 4.

FSC curves before (black) and after (red) 13-fold nonicosahedral averaging. The black curve suggests a resolution of 5.1 Å (0.143 threshold value), and the red curve indicates a resolution of 4.1 Å. By using the more conservative threshold of 0.5, the cut-off values are 6.5 Å (black curve) and 4.5 Å (red curve).

Fig. 5.

Correlation coefficients between images of DLPs and the reference. (a) Correlation coefficients are plotted as a function of particle and batch number (red, lacy carbon; green, C-flat). There is a bimodal distribution with a correlation coefficient of ≈0.14 separating the two clusters. (b) Correlation coefficients are plotted as a function of image defocus. (c) Correlation coefficients are plotted as a function of approximate particle distance from the center of hole in the carbon film.

Fig. 6.

Comparison of x-ray crystallographic and EM density maps. (a) X-ray crystallographic, icosahedrally averaged 2F_o − F_c density map at 3.8-Å resolution and atomic model built into this map (B. McLain, E.S., R.B., and S.C.H., unpublished data). The density corresponds to area 2 in Fig. 2c. (b) Same area as in a but showing the cryo-EM map. (c) Cryo-EM density in area 3 (Fig. 2c) showing clearly separated β-sheet strands. (d) Stereo image of area 4 (Fig. 2c) showing amino acid side chains protruding from different α-helices.