All refinements were performed with the gold standard procedure in RELION (Scheres and Chen, 2012). A post-processing procedure (Chen et al., 2013) was applied unless otherwise indicated. The dotted line is at FSC 0.143, used to determine the resolution from comparing two independently refined half data sets (Rosenthal and Henderson, 2003). (A) Comparison of different alignment procedures. Blue, average of 20 unaligned frames. Purple, 20 frames aligned with the statistical video processing procedure (Bai et al., 2013). Red, refinement after aligning frames with the area-motion correction software (Li et al., 2013). Gold, combination of the latter two alignment procedures. (B) Effect of radiation damage. Blue, 20 frames (3.69 Å); purple, 16 frames (3.69 Å); red, 12 frames (3.60 Å); gold, 8 frames (3.43 Å); green, 6 frames (3.39 Å). All curves were obtained by the combination approach described above. (C) Data set quality. Particle images of sub-standard quality were omitted from the original data set of 33,590 images, yielding a smaller dataset of 26,635 particles (‘Results’). Refinement using 8 frames of the reduced data set (green, 3.39 Å). The improvement is clear in comparison with the full dataset (blue, 3.43 Å). A further improvement (red, 3.36 Å) resulted from using 6 instead of 8 frames. (D) Post-processing to determine the resolution and B-factor (Chen et al., 2013) of the final map from the 6-frame refinement. The raw unmasked map (purple) indicates a resolution of 3.52 Å; the map masked with a soft mask (red) indicates the final resolution of 3.36 Å. The gold curve shows the FSC for the two half data sets with randomized phases beyond 4.5 Å. Subtraction of the gold curve from the red curve yields the green curve, which indicates the true map resolution corrected for over-aggressive masking. The close correspondence of the red and green curves shows that the used soft mask did not introduce spurious correlation and the true map resolution is 3.36 Å. A B factor of −156 Å2 was determined and applied to sharpen the map.