Typical RMSD plots for MD simulations before and after application of high [K⁺] show that the apo structure was equilibrated. After removal of Na⁺/Asp, the EAAT3-Glt_Ph homology model structure was used for MD runs in the 0.15 M K⁺ condition, with no binding events observed. In the first 5-ns period, a harmonic constraint of constant 0.04 was applied to the protein Cα. Constraints were then released, and the NPT simulation of the apo structure was allowed to equilibrate, resulting in an increase in RMSD due to a structural change of the aspartate-free structure. At the time indicated by the arrow, the K⁺ concentration was raised to 1 M. A K⁺ binding event was observed soon after, which led to only a slight change in RMSD.