Cryo-EM ribbon structures of the Ca ²⁺ -free human BK channel (PDB ID: 6V3G; Tao and MacKinnon, 2019) showing the location of the mutant arginine side chain G375R. (A) BK channel structure with the front and back pore-forming α subunits removed. The arginine side chain of the G375R mutation is shown in space-filling format on the back side of S6 away from the conduction pathway on the red-colored subunit. Carbon atoms are gray, nitrogen atoms are blue, and hydrogen is not indicated. (B) Structure after rotating 180° so that the location (blue mark) of the WT glycine side chain of G375 is visible on the back side of S6 on the yellow subunit. (C) Six possible orientations of the mutant arginine side chain are shown, generated by ChimeraX software (www.cgl.ucsf.edu/chimerax). The mutation G375D also produces a left shift in activation (G310D in Chen et al., 2014), but of half the magnitude of G375R (Fig. 3). This raises the possibility that the left shift may be related to the bulk of the substituted side chain rather than the specific charge, which is reversed for these two mutations.