Permeation mechanism in the E225A/E300A double mutant. Position of the incoming ion vs. the centroid of the two SF-bound ions located above it (left), and position of the leaving ion vs. the centroid of the two SF-bound ions located below it (right). Color bar is in units of log(ρ[x,y]); ρ[x,y] is the two-dimensional histogram of ion positions averaged over the entire simulation time of Sim. 26 and Sim. 27. Blue minima represent the four predominant three-ion and four-ion configurations in the knock-on permeation mechanism. All observed configurations are similar to those observed during permeation across the WT (see Fig. 1 E), indicating that the predominant permeation mechanism in this mutant is unchanged from that observed in the WT.