Extended Data Fig. 10: Cryo-EM analyses of the on-pathway 80S complexes during initiation and the transition to elongation.

a, Cumulative probability distributions of the dwell times for 60S joining to 48S PIC (that is, the formation of the 80S complex, which is shown in black and designated as ‘total 80S’) and the subsequent association of aa-tRNA with the A site (corresponding to the Δt value, which is shown in blue and designated as ‘80S EC’) were from the single-molecule experiment with the model mRNA and wild-type eIF5B at 20 °C and 3 mM free Mg²⁺ (n = 164). The red curve corresponds to the evolution of the fraction of the eIF5B-bound 80S complex (‘80S IC’) over time, simulated with the kinetics of the other two curves. The dashed lines indicate that at time point 45 s, around 64.5% of the 80S complexes should be in the 80S initiation complex state under these conditions. b, The population sizes of the two classes of 80S complexes that were observed by cryo-EM (80S IC, 17,602 particles, 70%; 80S EC, 7,542 particles, 30%) were in agreement with predictions from our single-molecule kinetics at time point 45 s. c–f, The compositional and conformational differences between the 80S initiation and elongation complexes. g. Summary of cryo-EM data collection and processing for the 48S PIC (Extended Data Fig. 2c–e), 80S initiation complex (c) and 80S elongation complex (d) maps.