(a) Transport cycle of GltPh consists of four steps. First, three Na+ ions and L-asp bind to the transporter in an outward-facing state (accessible from the extracellular side). Second, the transporter undergoes a conformational change from the outward- to an inward-facing state (accessible from the cytoplasm). Through this conformational change, Na+ ions and L-asp are translocated across the membrane. Third, the substrate and ions are released into the cytoplasm. Lastly, apo transporter undergoes a reverse conformational change back to the outward-facing state. (b) Residues from HP2 (red), HP1 (yellow), TM3, TM7, and TM8 (light blue) form the three Na+ binding sites (shown as pink spheres; based on PDB codes 2NWX and 5E9S) (Boudker et al., 2007; Guskov et al., 2016). The ions bind proximally to the substrate L-asp (shown in stick representation) but are not directly coordinated by it. (c) Structures of single GltPh protomers bound to L-asp in the outward- (left, PDB code 2NWX) and inward-facing states (right, PDB code 3KBC). Cα atoms of the cross-linked cysteine residues Cys55 and Cys364 and Hg2+ ion are shown as cyan and black spheres, respectively.