Figure EV1. Classification and refinement scheme for the pre‐40S complex, the 80S‐like particle, and the Dim1‐containing complex.

2D classification from 594,910 particles that had been picked from 2,875 micrographs revealed two particle subpopulations of “40S‐like” and “80S‐like” particles, which where separately processed and subjected to 3D classification and auto‐refinement yielding a 3D reconstruction of the pre‐40S complex at an overall resolution of 3.4 Å and a 3D reconstruction of the 80S‐like particle at an overall resolution of 3.1 Å. Subsequent rounds of focused 3D classification from this final set of pre‐40S particles focusing on either Rio2 (blue circle), Enp1 (red circle), or Dim1 (green circle) yielded subpopulations of the pre‐40S complex at overall lower resolution but with higher local resolution in the area of focus, further facilitating the model building and docking in these areas.