Supplementary Figure 4: Crystal structures of LeuT underestimate the closing of the extracellular vestibule.

Comparison of experimental distance distributions (solid lines) with predicted distributions based on the “inward-facing” (3TT3), “outward-facing” (3TT1) and “substrate-occluded” (2A65) crystal structures. The MMM package was used to generate predicted distance distributions on the extracellular side. This comparison demonstrates that the experimental distances in the Na⁺- and Leu-bound state fall outside the predicted distributions regardless of the crystal structure. Because MMM typically overestimates the distribution width, we interpret the systematic deviations between calculated and experimental distributions as evidence that the substrate-occluded conformation we observe in solution is not represented in the crystallographic record. Hence the crystal structures underestimate the closing of the extracellular side upon Na+ and Leu binding. The only exception to this seems to be EL4, for which the predicated distribution from the inward-facing structure overlaps the experimental distribution obtained in the presence of Na⁺ and Leu.