Fig. 4: A low energy barrier separates conducting and non-conducting states of the HERG selectivity filters.

A Representative structures of the two major clusters observed in constrained ion MD simulations. In Cluster 1 (blue, 41%) all selectivity filter backbone carbonyls point centrally. Cluster 2 (orange, 30%) has a flipped V625 backbone carbonyl that interacts with S620. B summary of distances between the indicated residue pairs for cluster 1 (blue) and cluster 2 (orange). Note that the S620 side chain is located ~2 Å from the Y616 backbone carbonyl ~5% of the time in cluster 1 and ~25% of the time in cluster 2. C–F Two-dimensional free energy surfaces obtained from REST2 simulations with MBAR for S620 side chain interactions with (C). V625 backbone carbonyl, D G626 backbone amide, E F627 backbone amide, and F Y616 backbone carbonyl, plotted as a function of rotation of the V625 backbone carbonyl angle (θ). Low free energy states are indicated for S620 interacting with (i) F627/G626, (ii) S620 interacting with Y616 backbone carbonyl when V625 backbone carbonyl points inwards, (iii) S620 interacting with Y616 backbone when V625 backbone carbonyl points outwards, and (iv) S620 interacting with flipped V625 backbone carbonyl. Qualitatively similar 2D PMF plots were obtained when we analyzed the amalgamated constrained ion simulations (see Supplementary Fig. S11).