Figure 6.

Work required by each D470-protonated simulation system to reproduce the experimental distance distribution. The work was computed by averaging the bias potential applied over the last 0.8 µs of each simulation of G450R5/S516R5 with Asp470 protonated. Simulations were performed with two bound sodium ions (2Na⁺), in either the absence (left) or presence (right) of betaine. The BetP conformation used to start the simulation was either inward- (PDB accession no. 4C7R, blue) or outward-facing (PDB accession no. 4LLH chain A, red). Error bars represent the SD of work values obtained by averaging over the two halves of the last 0.8 µs for the entire width of the distance distribution (A), or averaging the bias potential applied for distances <37 Å (B; see Materials and methods), as expected for exclusively inter-protomer interactions.