Fig. 2: Cryo-EM structure of rGAT1_EM in the cytosol-facing state.

a, Transport intermediates of GAT1 displaying cyclical inward- to outward-open states of the rGAT1. The GAT1 bound to GABA, a sodium at site 1 and a chloride ion is the primary conformation observed in the study (in color). b, Cryo-EM map of the refined rGAT1_EM structure (deep purple) bound to 9D5 (grey). The VH and VL part is ordered, although the constant domains lack clear density due to inherent disorder. c, The 12 transmembrane helix structure of rGAT1 in the detergent micelle (grey) displaying the transmembrane helices colored in spectrum. The VH (purple) and VL (pink) domains of the Fab were modeled and the constant domains were not modeled due to incomplete densities. N-glycosylation sites (NAG) and the disulfide bond are indicated in the extracellular loop 2. d, Electrostatic surface cutaway of rGAT1 displays substrate cavity exposed to solvent and the substrate GABA. The inset shows the thick extracellular gate and the hydrogen bond network of the residues that comprise the closed gate. Interaction distances are represented as dashed lines with values representing angstroms. e, The movement of TM1b and TM6a by 22° and 14°, respectively, obtained through structural comparison of an rGAT1 AlphaFold2 model in the outward-open state, allows closure of the extracellular gate. The inset shows the movement of Phe294 in the TM6 non-helical region that controls solvent access to the binding pocket and the side chain densities of Tyr140, Phe294 and Phe293 at σ level of 6.0.