Extended Data Fig. 2: Image processing scheme for the cryo-EM reconstruction of the 80S stalled complex on AUG-stop in +B conditions.

(a) Details of the image processing to obtain cryo-EM maps of the 80S stalled on AUG-stop in +B conditions and 40S initiation complexes with or without eIF2 binding. Of the 1,407 K particles initially 2D classified into 80S and 40S subgroups, 367 K particles sorted to 80S structures were auto-refined to obtain the consensus reconstruction. Particles were further 3D classified based on the alignment information from the consensus structure. Afterwards, 246 K particles were sorted to 80S ribosomes in complex with P-tRNA, E-tRNA, mRNA, and eRF1. The focused classification on eRF1 with residual signal subtraction was performed to obtain particles containing eRF1. The final reconstruction with 96 K particles was at a 2.9 Å resolution. In total, 181 K particles corresponding to 40S structures from the 2D classification were auto-refined to obtain the consensus reconstruction. 40S particles were 3D classified based on the alignment information from the consensus structure. Subsequently, 53 K particles were sorted to 40S ribosomes in complex with P-tRNA, mRNA, and eIF2. Focused classification on tRNA_i and eIF2 with residual signal subtraction was performed to classify particles into subgroups with or without eIF2 binding. As a result, 41 K particles without eIF2 and 12 K particles with eIF2 were reconstructed at 3.0 Å and 3.7 Å, respectively. (b-c) Resolution (gold-standard FSC) curves of three reconstructions (b) and models vs. cryo-EM maps (c) are shown. (d) Color-coded local resolution distributions of the cryo-EM map of the 80S complex from this dataset.