(a) Representative classes from RELION with 175 prescribed classes, and (b) the same as (a) except from P-RELION. (c) Representative classes from ISAC with 3X down-sampling, (d) the same as (c) except from P-ISAC (See Supplementary Figs. 15 and 16 for all the class averages). (e) The statistics of the total number of harvested particles. Six clustering experiments under different settings are conducted (colored with green/blue). 1: RELION/P-RELION with 175 classes, 2: RELION/P-RELION with 100 classes, 3: RELION/P-RELION with 50 classes, 4: ISAC without down-sampling, 5: ISAC/P-ISAC with 2X down-sampling, 6: ISAC/P-ISAC with 3X down-sampling. (f) The overall final resolutions corresponding to those in (e). (g) The map-to-model FSC curves by comparing the maps of RELION of 1, P-RELION of 1, ISAC of 6, and P-ISAC of 6 in (e) to PDB 3j5p. The calculation was performed using Phenix50. (h) The overall density maps of RELION of 1, P-RELION of 1, ISAC of 6 and P-ISAC of 6 in (e). The beta-sheets and the ankyrin repeats are indicated with a red arrow and a blue arrow, respectively. The B-factors measured and used for sharpening the four maps are 86.1,90.1,90.0, and 92.7, respectively. (i) enlarged densities corresponding to (h) where the same threshold of 0.49 on Chimera51 was applied. The left helix corresponds to 559–628 of subunit C, displayed as a reference. The middle and the right helices correspond to 429–455 and 469–498 of subunit A and they form a “U” with the connecting loop (456–468). The brown arrow denotes 464–468 while the green arrow denotes 456–458.