Fig. 3: Close up views of the dual receptor sites.

a Site IIB₂ (cyan) located between DIII (dark yellow) and DIV (dark red) interface. Amino acid residues that form the binding site are shown as sticks in brighter colors according to their domains (yellow for DIII and red for DIV). Two residues (lime green) in DII also contribute their main chain to the binding site. Part of BTX-B from Site IIB₁ (bright green) can be seen on the right. The stereo view of this receptor site with cryo-EM density map is shown in Supplementary Fig. 6a. Also see Supplementary Movie 1. b Close up view of the receptor site for Site IIB₁ (bright green) located between DI (dark blue) and DIV (dark red) interface. Amino acid residues that form the binding site are shown as sticks in brighter colors according to their domains (blue for DI and red for DIV). F399 and F403 in DI-S6 adopt the down rotamers in this model. In the structures of antiarrhythmic drug flecainide- and quinidine-bound rat and human Na_V1.5 (PDBs: 6UZ0 and 6LQA, respectively)^11,46, F399 and F403 both adopt an “up” rotamer that would clash with BTX-B. Furthermore, the F1667 side chain from DIV-S5 clearly adopts two partial rotameric conformations with equal distribution, one rotamer in the “down” conformation, closing down the interaction with the BTX-A moiety of the BTX-B site IIB₁, whereas the other rotamer in the “up” conformation makes no interaction with the bound BTX-B (Fig. 3b, Supplementary Fig. 6b, and Supplementary Movie 2). In all of the other Na_V1.5 structures mentioned above, F1667 uniformly adopts an “up” rotamer that points away from the BTX-B site IIB₁ suggesting that BTX-B binding favors the “down” rotamer. See also Supplementary Fig. 6.