Drift correction using all frames on 5 patches, dose-weighting, per frame local CTF estimation, as well as automated particle picking using a re-trained deep learning-based BoxNet method, were performed with Warp software [37]. Then, 2944 (for wild-type 30S subunits) and 3351 (for ΔrbfA 30S subunits) images under a 3.0 Å estimated CTF resolution threshold were used for further data processing, resulting in 489,736 and 514,927 particles, respectively.

All further data processing steps were performed using cryoSPARC v3.1.0/v3.2.0 software [38]. For both datasets, multiple rounds of reference-free 2D classification were performed using 50 classes at each step to minimize the number of false-positive particles in the subset and to remove non-30S subunit particles (50S subunits specifically, which represented less than 3% of particles in the case of ΔrbfA 30S subunit preparation). The results of heterogeneous ab initio reconstruction were used as an initial reference. In the case of wild-type 30S subunits, heterogeneous dataset refinement with 2 classes was performed to separate particles with a preferred orientation. Thus, for consensus refinement, 169,371 (for wild-type 30S subunits) and 352,365 (for ΔrbfA 30S subunits) particles were used, resulting in the maps of 3.2 Å and 2.7 Å resolution, respectively, estimated using an FSC = 0.143 gold-standard threshold. To analyze the structural heterogeneity of the sample, 3D variability analysis [39] with three main components was performed for both datasets. For ΔrbfA 30S subunits, several 3D classification strategies were tried. As a result, we settled on the clustering with 4 clusters in the reaction coordinate space using a Gaussian mixture model and then used these maps and two additional consensus maps as initial models for heterogeneous refinement. For each of the resulting classes, non-uniform refinement [40] was conducted. For wild-type 30S subunits and the particles of the most populated class F, local refinement was performed separately for the head and the body parts after CTF refinement and signal subtraction from the corresponding regions. For WT 30S subunits, a final local resolution was estimated as 3.1 Å for the body and 4.6 Å for the head, and for the class F intermediate—2.7 Å and 2.6 Å, respectively (Supplementary Materials Figures S2 and S3). All maps were locally filtered based on the local resolution estimation procedure implemented in the cryoSPARC software package.

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