Fig. 7: In situ cryo-ET, subtomogram averaging and 3D classification reveals that the majority of E.coli ED1a 70S ribosomes are in translation-incompetent state lacking P-tRNA after TET treatment.

a Digital slice from a representative tomogram of E. coli ED1a cells. The tomogram was denoised and the contrast was enhanced using IsoNet⁵⁹ trained on 5 tomograms with different defocus. Ribosomes appear as dark spherical objects. Scale bars 0.2 μm. b Three-dimensional view of the tomogram from (a) with aligned ribosome particles projected back into tomographic volumes. Small ribosomal subunits colored yellow, large subunits colored blue. Membranes segmented from tomogram are colored in gray. c The subtomogram average of the E. coli 70S ribosome from all datasets was resolved at 5.3 Å resolution. The structure represents an actively translating ribosome with mRNA (red), A-, P-, E-tRNA ligands, (pink, green, and beige respectively), elongation factors (EF, magenta) bound. d The solvent side view of the small ribosomal subunit of the overall subtomogram average of E. coli 70S ribosome. The enlarged inset shows details of the mRNA entry tunnel composed of universal r-proteins uS3, uS4, and uS5. The mRNA is supported by Lys44, and Arg46 of uS4, for which density is resolved to side-chain resolution. The segmented density for r-proteins shown in yellow, for mRNA in red. For visualization reasons, and to make the mRNA entry tunnel visible, the extra density at the polysome interface between the head and the body of the 30S is omitted. It can be seen colored in gray in figure (c). Subtomogram averages of the 70 S ribosome from (e) – E. coli K-12 (LB) at 8.5 Å, (f) – E. coli ED1a (LB) at 7.5 Å, (g) – E. coli K-12 (TET) at 8.5 Å, (h) – E. coli ED1a (TET) at 7.0 Å. The structure from the E. coli ED1a TET sample (h) shows only negligible density for tRNA and peptide exit tunnel (PET), suggesting the translation-incompetent state of most of the ribosomes in these cells. The 50S subunit is clipped to visualize the nascent chain inside PET (dark red), and the P-tRNA (green). Ribosomal subunits and ligands coloring: 30S – yellow, 50S – blue, mRNA – red, A-, P-, E-tRNA ligands – pink, green, and beige respectively, elongation factors (EF) – magenta. The distribution of ribosome classes sampled from E. coli K-12 30 min LB (i), E. coli K-12 30 min TET (j), E. coli ED1a 30 min LB (k), E. coli ED1a 30 min TET (l). Each segment of the pie chart represents a separate class obtained by 3D classification: 70S·P complex – green, 70S·EF-Tu·A/T,P,E complex – pink, 70S·EF-G·P,E complex – purple, 70S·P,E complex with weakly resolved elongation factor (EF) – gray, 70S·EF-Tu·A/T,E complex – light pink, 70S·EF-G·E complex – orchid, 70S·E complex with weakly resolved elongation factor (EF) – light gray. The thick green contour defines ribosome classes of translation-competent ribosomes charged with P-tRNA, the thick red dashed contour defines classes of translation incompetent ribosomes lacking P-tRNA. The thin black contours outline structurally similar 3D classes. Number of particles for each class is shown next to corresponding sector. Source data are provided as a Source Data file.