Fig. 2: Cellular cryo-ET of 5-nm-HN-labeled ribosome.
a, Schematic of live-cell delivery of 5-nm-HN, vitrification, cryo-FIB milling, cryo-ET and tomographic reconstruction to generate 3D volumes. b, Schematic of a 5-nm-NH-labeled ribosome. The white dot represents the C terminus of L29 that defines \(\hat{r}\)R, and the center of the nanogold defines \(\hat{r}\)G. c, Central slice (8 Å per pixel) of an individual ribosome labeled with 5-nm-NH by weighted back-projection (contrast is as in original data, where dark pixels correspond to high occupancy, that is, black-on-white), the same subtomogram deconvolved, its corresponding normalized subtomogram and the subtomogram average from 13,748 ribosomal picks, dominated by alignment of nanogold intensity (contrast inverted, that is, white-on-black). Experiments were repeated twice independently with reproducible results. d, Central slice of a weighted back-projection of the whole tomogram, averaged over ten slices. e, Violin plot of the pairwise distances between ribosomal subunit L29 (rR) and 5 nm-HN (rG), mean = 5.1 nm, s.d. = 1.9. N = 9,567 pairwise distances. f, Bar graph of the labeling efficiency (0.695) and how the labeling efficiency was calculated. Scale bars, 25 nm (b); 25 nm (c); 20 nm (d).
