Figure 1: LeuT in the outward-oriented and Na⁺–free state shows relocation of conserved Leu25 into the substrate-binding site.

(a) Electron density maps for TM1 after MR (left) showing unbiased indications for Leu25 relocation (final model and maps, right). 2F_o-F_c maps are shown at 1 r.m.s.d. (grey mesh) and unbiased F_o-F_c maps at 3.5 r.m.s.d. (green mesh positive, red mesh negative). (b) The outward-oriented, Na⁺-free state (pink, this study) superimposed on the Na⁺- and L-leucine-bound, occluded outward-oriented state³ (grey, PDB entry code 2A65). Leu25 overlaps with the substrate-binding site. (c,d) Outward-oriented Na⁺-free state (pink, this study) superimposed with Na⁺-bound outward-open (c, grey, PDB entry code 3TT1) and Na⁺-free inward-open (d, sky blue, PDB entry code 3TT3) states². (e) Molecule A cartoon putty thickness illustrating flexibility and colour gradient for atomic displacement B-factor from blue (thin, low disorder) to red (thick, high disorder) with an average value of 93.4 Ų. (f) Alignment of the TM1 region of NSS. The conserved Leu25^LeuT is marked with a red box. MR, molecular replacement.