Extended Data Fig. 9: Time series of Ca²⁺ position in simulations with D153 and D471 deprotonated or protonated.

Plots are shown for five independent simulations under each of four conditions: (1) simulations of cytosol-open NCLX initiated with a Ca²⁺ ion in the binding pocket, with D153 and D471 deprotonated (blue traces); (2) simulations of cytosol-open NCLX initiated with a Ca²⁺ ion in the binding pocket, with D153 and D471 protonated(gray traces); (3) simulations of matrix-open NCLX initiated with a Ca²⁺ ion in the binding pocket, with D153 and D471 deprotonated (orange traces); (4) simulations of matrix-open NCLX initiated with a Ca²⁺ ion in the binding pocket, with D153 and D471 protonated (brown traces). The z-axis is the direction perpendicular to the lipid bilayer. The z-coordinate is 0 at the initial Ca²⁺ position, with values increasing in the direction of the matrix and decreasing in the direction of the cytosol. Red arrows mark times at which the Ca²⁺ exits the transporter; subsequently, it either interacts with charged residues at the protein surface or diffuses freely through the solvent. Unsmoothed traces (thin lines) and smoothed traces (thick lines) are shown for all simulations. Time traces were smoothed using a moving average with a window size of 20 ns.