Structure of two opposing subunits of the KcsA tetramer representing the closed state (a), the partially open and fully open states (b) and, the inactivated state (c). Potassium cations are represented by purple spheres, membrane lipid headgroups by white spheres, and bound DOPG lipids by their molecular surface in red. The typical values for their inner gate (IG) and selectivity filter (SF) openings are shown below each snapshot. (d) Representative MD snapshot of the pathway taken by water molecules before the final step of inactivation. This pathway passes between L81 and W67 (green dashed line) allowing for two additional water molecules to be buried behind the selectivity filter.

Reweighted free energy surfaces calculated from the string method with swarms of trajectories. 2D free energy surfaces projected on the inner gate distance (IG, average cross-subunit distance between the CA of T112) and the selectivity filter distance (SF, average cross-subunit distance between the CA of G77 residues) of the systems using (a) AMBER and (b) CHARMM. 1D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) of the systems using (c) AMBER and (d) CHARMM with labels denoting the state of the basin: closed (C), partially open (PO), fully open (FO) and inactivated (I). The errors associated with the 2D-free energy surface can be found in Figure 2—figure Supplement 4

Figure 2—figure supplement 1. Evolution of the string beads over the string method iteration (averaged over blocks of 25 iterations) and projected on the inner gate distance (IG, average cross-subunit distance between the CA of T112) and the selectivity filter distance (SF, average cross-subunit distance between the CA of G77 residues). The initial string is represented as a black dotted line. The inset plots the first ten iterations without averaging. The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Figure 2—figure supplement 2. Evolution of the root mean squared deviation from the initial string averaged over all beads. The systems represented are LB-AMBER (orange), LB-CHARMM (blue) and noLB-AMBER (red).

Figure 2—figure supplement 3. The weighted average value of 𝑠𝑝𝑎𝑡ℎ projected 2D-free energy surface projected on the IG and SF. The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Figure 2—figure supplement 4. Estimation of the uncertaintly associated with the 2D-free energy surface projected on the IG and SF (see Methods). The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Figure 2—figure supplement 5. IG and SF projections of 200 ns MD trajectories started from representatives of different states of the inactivation cycle onto corresponding free energy surface. The starting structure is represented as a triangle. The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the AMBER with lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interest are: (a) Inner gate distance, (b) T74 CA - F103 CA distance, (c) E71 𝜒1 Janin angle, (d) F103 𝜒1 Janin angle and (e) R64 CZ - D80 CG distance. (f) Projected weighted average number of water molecules inside the selectivity filter (sites S1-S4) on the free energy surface of the AMBER lipid bound system.

Figure 3—figure supplement 1. Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the AMBER with lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interest are: (a) T74 CA - I100 CA distance and (b) R89 CZ - D80 CG distance (c) Copurifying DOPG P - R64 CZ distance (d) Copurifying DOPG P - R89 CZ distance.

Figure 3—figure supplement 2. Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the CHARMM with lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interest are: (a) Inner gate distance, (b) T74 CA - F103 CA distance, (c) E71 𝜒1 Janin angle, (d) F103 𝜒1 Janin angle, (e) R64 CZ - D80 CG distance, (g) T74 CA - I100 CA distance and (h) R89 CZ - D80 CG distance. (f) Projected weighted average number of water molecules inside the selectivity filter (sites S1-S4) on the free energy surface of the CHARMM lipid bound system (i) Copurifying DOPG P - R64 CZ distance (j) Copurifying DOPG P - R89 CZ distance.

Figure 3—figure supplement 3. Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the AMBER without lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interes9toafre1:9(a) Inner gate distance, (b) T74 CA - F103 CA distance, (c) E71 𝜒1 Janin angle, (d) F103 𝜒1 Janin angle, (e) R64 CZ - D80 CG distance, (g) T74 CA - I100 CA distance and (h) R89 CZ - D80 CG distance. (f) Projected weighted average number of water molecules inside the selectivity filter (sites S1-S4) on the free energy surface of the AMBER without lipid bound system (i) Copurifying DOPG P - R64 CZ distance (j) Copurifying DOPG P - R89 CZ

Reweighted free energy surfaces calculated from the string method with swarms of trajectories. 2D free energy surfaces projected on the inner gate distance (IG, average cross-subunit distance between the CA of T112) and the selectivity filter distance (SF, average cross-subunit distance between the CA of G77 residues) of the system with (a) PG-lipids bound and (b) no PG-lipids bound using the AMBER force field. 1D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) of the system with (a) PG-lipids bound and (b) no PG-lipids bound using the AMBER force field with labels denoting the state of the basin: closed (C), partially open (PO), fully open (FO) and inactivated (I).

Schematic depiction of the two possible inactivation mechanisms of KcsA as described by the minimum free energy paths obtained by the string method with swarms of trajectories for the AMBER (a) and CHARMM (b) force fields.

Distance CVs used for the steering simulations. AB and CD subunits are opposing each other and AC, CB, BD and DA subunits are side by side in a clockwise fashion observing the channel from the intracellular side.

Distance CVs used for the string simulations. AB and CD subunits are opposing each other and AC, CB, BD and DA subunits are side by side in a clockwise fashion observing the channel from the intracellular side.

Evolution of the string beads over the string method iteration (averaged over blocks of 25 iterations) and projected on the inner gate distance (IG, average cross-subunit distance between the CA of T112) and the selectivity filter distance (SF, average cross-subunit distance between the CA of G77 residues). The initial string is represented as a black dotted line. The inset plots the first ten iterations without averaging. The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Evolution of the root mean squared deviation from the initial string averaged over all beads. The systems represented are LB-AMBER (orange), LB-CHARMM (blue) and noLB-AMBER (red).

The weighted average value of 𝑠𝑝𝑎𝑡ℎ projected 2D-free energy surface projected on the IG and SF. The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Estimation of the uncertaintly associated with the 2D-free energy surface projected on the IG and SF (see Methods). The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

IG and SF projections of 200 ns MD trajectories started from representatives of different states of the inactivation cycle onto corresponding free energy surface. The starting structure is represented as a triangle. The systems represented are (a) LB-AMBER, (b) LB-CHARMM and (c) noLB-AMBER.

Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the AMBER with lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interest are: (a) T74 CA - I100 CA distance and (b) R89 CZ - D80 CG distance (c) Copurifying DOPG P - R64 CZ distance (d) Copurifying DOPG P - R89 CZ distance.

Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the CHARMM with lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interest are: (a) Inner gate distance, (b) T74 CA - F103 CA distance, (c) E71 𝜒1 Janin angle, (d) F103 𝜒1 Janin angle, (e) R64 CZ - D80 CG distance, (g) T74 CA - I100 CA distance and (h) R89 CZ - D80 CG distance. (f) Projected weighted average number of water molecules inside the selectivity filter (sites S1-S4) on the free energy surface of the CHARMM lipid bound system (i) Copurifying DOPG P - R64 CZ distance (j) Copurifying DOPG P - R89 CZ distance.

Reweighted free energy surfaces calculated from the string method with swarms of trajectories of the AMBER without lipids bound system. 2D free energy surfaces projected on the path cv (𝑠𝑝𝑎𝑡ℎ) and an additional CV of interest. These CVs of interest are: (a) Inner gate distance, (b) T74 CA - F103 CA distance, (c) E71 𝜒1 Janin angle, (d) F103 𝜒1 Janin angle, (e) R64 CZ - D80 CG distance, (g) T74 CA - I100 CA distance and (h) R89 CZ - D80 CG distance. (f) Projected weighted average number of water molecules inside the selectivity filter (sites S1-S4) on the free energy surface of the AMBER without lipid bound system (i) Copurifying DOPG P - R64 CZ distance (j) Copurifying DOPG P - R89 CZ distance.

W67-L81 distance strings as a function of the string method iteration number for the AMBER system and with each graph representing the data for a particular subunit of the tetramer.

W67-L81 distance strings as a function of the string method iteration number for the CHARMM system and with each graph representing the data for a particular subunit of the tetramer.

W67-D80 distance strings as a function of the string method iteration number for the AMBER system and with each graph representing the data for a particular subunit of the tetramer.

W67-D80 distance strings as a function of the string method iteration number for the CHARMM system and with each graph representing the data for a particular subunit of the tetramer.

Projected weighted average number of water molecules (blue) or potassium ions (purple) inside a particular selectivity filter sites, S1 to S4 (rows), on the IG vs SF free energy surface of the AMBER with lipid bound system.

Projected weighted average number of water molecules (blue) or potassium ions (purple) inside a particular selectivity filter sites, S1 to S4 (rows), on the IG vs SF free energy surface of the CHARMM with lipid bound system.

Projected weighted average number of water molecules (blue) or potassium ions (purple) inside a particular selectivity filter sites, S1 to S4 (rows), on the IG vs SF free energy surface of the AMBER without lipid bound system.