MD simulations of ion permeation through the structural model of the disulfide-reduced GLIC C27S + K33C + I9′A + N21′C mutant. The membrane was bathed by symmetrical 150 mM KCl, and the temperature was 37°C. (A) Ion trajectories at −100 mV. (B) Ion trajectories at −200 mV. In A and B, only the trajectories of ions that crossed the membrane are plotted. In these plots, gray areas indicate the regions occupied by the membrane in the periodic-simulation system, and downward transitions of the ion trajectories through these regions represent inward crossings. The darker lines are running averages of the data, which are displayed in a lighter shade. The length of the simulation box along the z-axis was 108 Å. (C) Pore-radius profiles (estimated using HOLE [Smart et al., 1996]) of structural models of the disulfide-reduced quadruple mutant. The profile of the crystal-structure model (PDB ID code 5V6N; this work) is compared with those computed during the ion-permeation MD simulations illustrated in A and B. (D) Current–voltage relationship from simulations performed at −100, −200, and −500 mV; ion trajectories at −500 mV, and the corresponding MD pore-radius profile, are shown in Fig. S2. (E) Pore-radius profiles of structural models of the closed-channel conformation. The profile of the crystal-structure model (PDB ID code 4LMK; Gonzalez-Gutierrez et al., 2013) is compared with that computed during an ion-permeation MD simulation at −100 mV. In C and E, ion-permeation-MD pore-radius profiles are mean profiles—displayed as the mean (darker lines) ± 1 SD (lighter shade)—calculated from the different frames of each simulation; the vertical axes extend, approximately, between M2 positions −3′ (bottom) and 21′ (top). Side-chain rotamers were optimized using SCWRL4 (Krivov et al., 2009) before the ion-permeation simulations were run.