Extended Data Fig. 6: Image processing scheme of the cryo-EM reconstruction of the 80S stalled complex on AUG-stop in +B/ − B conditions with CHX.

(a) Details of image processing to obtain the cryo-EM map of the 80S stalled on AUG-stop in +B conditions with CHX addition. First, 131 K particles were initially 2D classified to obtain 80S particles. The 98 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. 80S ribosomes in complex with P-tRNA, mRNA, eRF1, and CHX were selected. Focused classification on eRF1 with residual signal subtraction was performed to obtain particles containing eRF1. The final reconstruction with 69 K particles was at a 3.4 Å resolution. (b) Details of image processing to obtain the cryo-EM map of the 80S stalled on AUG-stop in −B conditions with CHX addition. The picked 220 K particles were initially 2D classified to obtain 80S particles, and then 92 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. 80S ribosomes in complex with P-tRNA, mRNA, and CHX were selected. The focused classification on the fragmented eRF1 density with residual signal subtraction was performed. The obtained 52 K particles containing some density on the A site were reconstructed at a 3.4 Å resolution. (c-d) Resolution (gold-standard FSC) curves of two reconstructions (c) and models vs. cryo-EM maps (d) are shown. (e-f) Color-coded local resolution distributions of the cryo-EM map of the 80S complex from these datasets.