Extended Data Fig. 3: Cryo-EM data processing and particle classification workflow (dataset II with GDPCP).

All steps were performed in cryoSPARC 3.3.2⁷⁸. 9,810 micrographs were collected, of which 9,390 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 334,113 particles (class average I), focused 3D variability analysis around RF3 and RF1 was utilized to separate 31,256 particles for containing solid density for RF3 and RF1 or for RF1 only (146,778 particles). The process discarded 59,038 particles containing non-rotated ribosomes with weak density for RF3 and RF1, 58,392 particles containing non-rotated ribosomes with no factor bound, 8,460 particles of non-rotated ribosomes with only RF3 bound, and 30,189 particles that yielded noisy reconstructions. Non-uniform and CTF refinement of particles with RF1 and RF3, or with RF1 only, produced structures II-A and II-D, respectively. Local refinement was combined with particle subtraction to produce a higher-quality map of RF3 and RF1 in structure II-A. Similarly, the rotated ribosome particles (186,545 particles in class average II) were separated by focused 3D variability analysis around RF3, producing two defined volumes (56,202 and 116,992 particles), both of which containing RF3 in a distinct binding position. Non-uniform and CTF refinement of the latter two volumes yielded structures II-B and II-C. Local refinement combined with particle subtraction produced higher-quality maps of RF3 in structures II-B and II-C.