Extended Data Fig. 4: Classification of ribosomes translation elongation states in Cm-treated M. pneumoniae cells.

a, The cryo-ET data processing and sub-tomogram classification workflow. At least three tiers of classifications were performed, including global 70S classification, focused classification on A-P-E-tRNA association sites (mask I), and on elongation factor and A/T-tRNA binding region (mask II). For each class, the average map, a class name, particle numbers and the global resolution (FSC = 0.143) are provided. Two classes generated at the first tier of global 70S classification (769 and 1,177 particles) resulted in low-resolution ribosome maps with unexplainable densities and are not shown. These are denoted as ‘unclear’ in Extended Data Fig. 5. b, Local resolution maps (color coded according to the bar on bottom left) for the six major classes. c, Local resolution map (color coded according to the bar on bottom right) for the minor class. d-f, FSC curves for all classes based on RELION postprocessing. g-h, In the ‘a, P’ and ‘a, P, E’ classes, the A-site tRNA’s anticodon end bound to the 30S decoding center is well-resolved (yellow arrowhead), but the body shows blurred density. They possibly represent 70S intermediates with the incoming tRNA sampling between the A/T- and the classic A-site (as illustrated in h). Maps were low-pass filtered to 6 Å for comparison. i, The ‘A*, P/E’ minor class represents intermediates between the pre-translocational hybrid H1 and H2* states²⁵. Models were fitted in the map for comparison. j, Comparison of the distribution of the classified translation elongations states of 70S ribosomes in untreated¹⁵ and Cm-treated cells.

Source data