For model building, the 30S subunit (chain A) was extracted from a E. coli vacant 70S ribosome crystal structure model (PDB ID 4V4Q). Head and body domains of the 30S subunit were fitted into cryo-EM maps as a rigid body using UCSF Chimera software [41]. The initial course fitting of flexible elements into the density was performed using the Namdinator web service (https://namdinator.au.dk (accessed on 8 March 2021)). The default parameters were used for flexible fitting. Models and maps were visually inspected in the Coot program [42]. The final models were built after several rounds of real space refinement in the Phenix program suite [43] and manual model building in Coot. For model validation, we used MolProbity from Phenix. Data and refinement statistics are summarized in Supplementary Materials Table S1.

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