Atomistic MD simulations

For simulations of KdpFABC, the structural model of 7NNL was truncated by removing the KdpB soluble domains in the regions KdpB_V100-G196 and KdpB_D302-L558. Positional restraints (1000 kJ mol⁻¹ nm⁻²) were applied to backbone atoms C, C_α, and N to prevent shifts in the input structure. Alternatively, simulations were built using the full-length KdpFABC in the E2 state 7BGY. Protein atoms were described using the CHARMM36 force field, and inserted into a 67% POPE, 23% POPG, 10% CL lipid bilayer solvated with TIP3P water and 150 mM K⁺/Cl⁻ using CHARMM-GUI^82–84. For the majority of the simulations, the protonation states of all side chains were set to default apart from KdpB_K586, and KdpA_E370, which had pKas of 7.5, and 9.1, respectively, based on analysis with PropKa3.1⁸⁵. Additional systems were built in which KdpB_D583 and KdpB_K586 were either both charged or both neutral, as well as a neutral KdpB_E370 (Supplementary Fig. ^{14a, b}).

Systems were energy minimized using the steepest descents method, and subsequently equilibrated with positional restraints on heavy atoms for 100 ps in the NPT ensemble at 310 K with the V-rescale thermostat and semi-isotropic Parrinello-Rahman pressure coupling^80,81. For both stages, positional restraints of 1000 kJ mol⁻¹ nm⁻² were applied to the K⁺ in the tunnel. Production simulations were run in triplicate with 2 fs time steps for ca. 250 ns unless specified.

Ion density analyses were run using the VolMap tool of VMD⁸⁶ with the default settings, taken over 3 × 250 ns of simulation for each system.

All atomistic and CGMD simulations were run in Gromacs 2019⁸⁷.