Extended Data Fig. 1: Cryo-EM data processing and particle classification workflow (dataset I with RF3-GDP and no exogenous nucleotide).

Scheme of the data processing workflow. All steps were performed in cryoSPARC 3.3.2⁷⁸. 10,284 micrographs were collected, of which 8,923 were selected for further processing. After two rounds of reference-free 2D classification, the selected particles were used to generate ab-initio volumes. Particles from the 3D volumes that appeared to be the 70S ribosome based on their shape and size were separated according to their ratcheted state using heterogeneous refinement, yielding three main classes of particles (class averages I, II, and III). Among the non-ratcheted 326,675 particles (class average I), focused 3D variability analysis around RF3 and RF1 was utilized to separate 80,766 particles for containing solid density for RF3 and RF1 or for RF1 only (127,567 particles). The process discarded 40,336 particles containing non-rotated ribosomes and 12,965 particles of rotated ribosomes, both of which were not bound to any factor. The remaining 65,041 particles yielded highly noisy reconstructions and were not further analyzed. Non-uniform and CTF refinement of particles with RF1 and RF3, or with RF1 only, produced structures I-B and I-A, respectively. Local refinement was combined with particle subtraction to produce a higher-quality map of RF3 and RF1 in structure I-B. Similarly, the rotated ribosome particles (74,452 particles in class average II) were separated by focused 3D variability analysis around RF3, producing two defined volumes, one containing rotated ribosome with no factor bound (28,078 particles) and one with rotated ribosome bound to RF3 (33,348 particles). Non-uniform and CTF refinement of the latter volume yielded structure I-C. Local refinement combined with particle subtraction produced a higher-quality map of RF3 in structure I-C.