Fig. 1: Cryo-EM structure of WT-HERG solved in presence of high-K and low-K.

A Cryo-EM map of low-K WT-HERG (contour 6σ) with a single subunit of the channel highlighted to illustrate the major domains: pore-domain (PD), voltage sensor domain (VSD), cyclic nucleotide binding homology domain (cNBHD), per-arnt-sim (PAS) domain. B Local resolution of cryo-EM maps solved using C4 symmetry. The resolution range of the transmembrane regions are reported in Supplementary Table 1. C Fractional difference map for high-K and low-K maps. Maps were normalized and low pass filtered to 3.3 Å before fractional difference was calculated using TEMpy:Difference Map tool in CCPEM⁵³. Red indicates density only present in the low-K map (density missing in high-K) and blue represents density only present in the high-K map; thresholding 0.0252 (8σ). Right panel shows a close-up view of the selectivity filter (high-K, blue and low-K, red). In addition to the selectivity filter residues, there are differences seen for the S620 and Y616 side chains. D overlay of low-K (orange) and high-K selectivity filter structures, aligned to the pore helix. In the left panel the lateral and upward displacement of the F627/G628 backbone in the low-K structure is apparent as is a small lateral displacement of the S624 side chain. On the rotated view, the central displacement of the G626 backbone carbonyl, flipping of the V625 backbone carbonyl (bidirectional arrows) and resulting central facing G626 amide (arrowhead) are apparent in the low-K structure. E The high-K structure has a canonical selectivity filter HOLE profile, whereas the low-K structure shows dilation in the vicinity of V625 and constrictions at the level of S624 and G626. This is distinct to the single constriction observed in the KcsA low-K filter at G77 (equivalent to G626, see Supplementary Fig. S6). F Example MD simulations showing ion conduction events when the V625 backbone carbonyls point towards the central cavity (to mimic high-K structure), but no conduction is seen when the V625 backbone carbonyls point outwards (to mimic low-K structure). Ions are shown as colored lines, water molecules as gray points.