Extended Data Figure 1. In silico 3D classification scheme.

a) All particles were subjected to 2D classification, from which non-ribosomal particles were discarded, prior to 3D refinement. To isolate particles containing A-site tRNA and RelA, 3D classification focused on occupancy of the ribosomal A-site was performed. Refinement of these 183,615 particles resulted in a reconstruction with a nominal resolution of 2.9 Å. A second round of 3D classification isolated 164,353 well-aligned particles. Conformational heterogeneity of the ribosome was resolved by 3D classification without alignment, which identified two dominant classes in which the body of the small subunit occupies different positions (indicated with an arrow). Class 1 was used as the reference for model building, refinement, and interpretation. To resolve additional conformational heterogeneity of RelA, focused classification with signal subtraction (FCwSS) was performed on each domain, with the hydrolase (HYD) and synthetase (SYN) domains treated as a single unit. For the RRM, ZFD and TGS domains a single class was isolated in which the density was better resolved than in the reference class. The overall resolution of the reconstructions are reported according to the FSC=0.143 criterion. Multiple conformations of the HYD and SYN domains were identified, with the four best resolved classes shown. Together these account for 42% of the particles. b, The two main classes for the TGS domain provide an example of the small conformational differences that were isolated using FCwSS.