(A) Histogram of tilt-pair alignment errors for particles that were calculated as 16-frame averages. The width of the first peak at half its height is 2°. This value is plotted as the tilt-pair alignment precision in (B). (B) Tilt-pair alignment precision (top) and the number of incorrectly aligned particle pairs (bottom) for independent refinements of 16-frame, 8-frame, 6-frame, 4-frame and 2-frame averages (ranges in frame numbers are indicated on the x-axis); the total number of particle pairs was 15,202. Particle pairs with alignment errors larger than three times the reported precision were considered as aligned incorrectly. (C) Gold-standard FSC curves. The same blue colors are used as in (B); orange lines indicate the results of the statistical video-processing approach described in the main text. (D) FSC-curves between a rigid-body fitted atomic model and the cryo-EM maps (using the same color scheme as in [C]). (E–F) Illustrative density and atomic model for the reconstructions obtained from 16-frame averaged particles (light blue), independently refined four-frame averages (dark blue) and the statistical video processing procedure (orange). The density maps were sharpened with B-factors of −211, −185 and −178 Å2, respectively. Complete density maps for these three reconstructions are shown in Figure 2—figure supplement 1.