Distributions of K⁺ and amine energetics of SPD³⁺ in the MthK pore. (A) Fractional occupancy of the bottommost K⁺ ion versus position along the pore axis from simulations with SPD³⁺, at 0 mV (top row). Panels in the middle row contain the free energy profiles for the N_L, conditioned on the position of the bottommost K⁺ ion, superimposed on histogram of the corresponding K⁺ occupancy. Snapshots of structures corresponding to the global minima of the free energy plot are shown below; two of the MthK pore-forming subunits are shown for clarity (light blue); SF main chain, polyamine, and SF-bound water molecules are shown as sticks, and SF-bound K⁺ ions are shown as purple spheres. (B) Corresponding histograms, free energy profiles (middle), and structure snapshots from simulations with SPD³⁺, at 500 mV. Whereas conditional free energy profiles are similar with 0 or 500 mV, the bottommost K⁺ ion is driven from the pore cavity toward the SF with depolarization, thus driving N_L toward increased occupancy at the entry to the SF, to occlude K⁺ permeation. Dashed lines in A and B correspond to SEM, computed by considering points from the variable sampled every 1 ns.