Data S1, Related to Figure 120S proteasome (EMPIAR 10025) and -galactosidase (EMPIAR 10061) refinement strategy and analysis(A) Representative reference-free 2D class averages showing the quality of 2D averages that were selected for 3D analysis of 20S proteasome. (B) 20S particles from selected 2D class averages were subjected to gold-standard 3D refinement. Following particle polishing and per-frame weighting, the refined “shiny” particles were subjected to a classification without alignment. The class containing the highest resolution features was then further refined to yield the final 2.6 Å resolution map. (C) Estimated per-frame B-factor (top) used for per-frame radiation damage weighting of 20S proteasome (bottom), as estimated by the RELION particle polishing routine. First frame is colored in yellow, and the frame with the least negative relative B-factor (Bf) is colored in orange (frame 5) (D) Fourier shell correlation (FSC) plot calculated from independently refined half-maps of 20S proteasome. 0.143 FSC cutoff is represented as a gray line. (E) Euler distribution of the final 20S proteasome density. (F) Representative reference-free 2D class averages of -galactosidase. (G) -galactosidase particles from selected 2D class averages were subjected to gold-standard 3D refinement. Following particle polishing and per-frame weighting, the refined “shiny” particles were subjected to a no-alignment classification. The best class was then further refined to yield the final 2.2 Å resolution map. (H) Estimated per-frame B-factor (top) used for per-frame radiation damage weighting of -galactosidase (bottom). (I) Fourier shell correlation (FSC) plot calculated from independently refined half-maps of -galactosidase. 0.143 FSC cutoff is represented as a gray line. (J) Euler distribution of the final -galactosidase density.