Supplementary Figure 3: Quality of EM density map.
From: Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2)
EM density maps are shown for (a) the entire monomer, (b) 4 TM helices (S1-S4; cyan) of the VSLD, (c) helices (S4L, S5, PH, S6; blue) in the pore domain, (d) external helices (α1-3; orange) in the TOP domain, (e) central β sheet of TOP domain along with density for individual strands, (f) three glycosylation sites in the TLC motif of the TOP domain (NAG3-5). The EM density map shown corresponds to the final RELION reconstruction filtered at 4 Å and sharpened with a B-factor of –150 Å2. This sharpened map was used also used as input to REFMAC-EM for model refinement in reciprocal space. The map (dark blue mesh) is contoured at 3.5σ. For the glycosylation sites shown in (f), the unsharpened EM map, contoured at 3.5σ is also shown (cyan mesh). Bulky amino acids that aided sequence assignment and register are highlighted. (g) 8.5 Å Anomalous difference Fourier map calculated from the crystallographic data collected from the EMTS-soaked crystals contoured at 3σ. The map (magenta mesh) is overlaid on the ribbon trace of a PC2 monomer with the positions of the nine cysteine residues highlighted. Hg peaks corresponding to all cysteines in the TM helices are observed. (h) Overall view of PC2 tetramer showing the unsharpened 4.2 Å EM density map. Dotted region indicates characteristic C-terminal ‘bulb’ at end of S6B helices. (i) Final refined model with overlaid unsharpened map contoured at low (4.3σ) level. (j) Possible extension of S6B helix has been modelled along with an additional short C-terminal helix. Density for the helical portions is resolved but the connecting regions are not. (k) Density between S6, pointing towards the pore axis, is compatible with a short helix.
