Supplementary Figure 2: Single-particle cryo-EM of human PC2.

(a) Representative raw particles from an original micrograph movie stack after initial frame alignment and summation. (b) Reference-free 2D class averages of detergent solubilised PC2 particles. (c) Initial ‘spinning top’ 3D model generated from a side-on 2D class average used for initial rounds of 3D classification. (d) Resolution estimation based on the gold standard FSC curve from RELION. The FSC curves for both unmasked and masked models calculated using RELION’s automatic post-refinement masking procedure are shown. The overall resolution is calculated to be 4.22 Å based on FSC 0.143. (e) FSC curve of the final 4.2 Å REFMAC-EM refined model versus the map it was refined against (FSC_full, black line). For the purposes of validation, the (semi-randomised) model was refined against a reconstruction generated from half of the particles and then this refined model was compared to an independent reconstruction generated using the remaining particles. The FSC curves are shown for the unbiased model refined against the first of two independent (half) maps (to which it was refined against; FSC_work; red line) or the same refined model versus the second independent half map (to which is was not refined; FSC_half2, green line). (f-g) Local resolution variation throughout the map estimated by RESMAP. Local resolution variation is color-coded from 4-6 Å. (f) Map contoured at low level to indicate the disordered detergent micelle ring covering the TM regions and the partially ordered C-terminal region. (g) Map contoured at higher level showing the PC2 tetramer viewed in the membrane plane (left) and two perpendicular slices through the density at the level of the TOP domain and around the pore. The central helices surrounding the pore and the core elements of the TOP domain exhibit resolutions around 4 Å. The cytosolic N- and C-termini as well as the S2-S3 and S4-S4L loop regions and the extremities of the TOP domain are less well ordered.