(a) Representative classes of NC-TRPV1 from RELION with 200 prescribed classes and (b) from P-RELION. (c) Representative classes from ISAC with 3X down-sampling on NC-TRPV1, (d) from P-ISAC (See Supplementary Figs. 15 and 16 for all the class averages). For (e)–(g), from left to right are the results of RELION, P-RELION, and P-RELION (second pass). (e) Representative classes of NanoD-TRPV1 using RELION or P-RELION with 100 prescribed classes (See Supplementary Fig. 17 for all the class averages), (f) the FSC resolution for NanoD-TRPV1, (g) shows the overall density maps (threshold = 0.55). The B-factors measured and used for sharpening the three maps are 130.6, 123.5, 117.3, respectively.