Extended Data Fig. 9: Structural comparison among members of SSS family.

a, Superposition of the binding pockets of SMCT1 and apo SGLT1_conHA. Transmembrane helices involved in forming the central pockets are labeled. The relative positions of substrates are indicated by 3D shapes: pink hexagon for glucose and orange oval for butyrate. SMCT1 and SGLT1_conHA are colored in cyan and gray, respectively. b, Surface representation of the central cavity of SMCT1 (left) and SGLT1_conHA (right). Substrates are placed in the binding sites, the extension of which is indicated by dashed yellow ovals. c, The sequence comparison of the substrate binding site residues. For the sugar-transporting branch, the positions equivalent to SGLT1’s sugar binding site residues are highlighted in orange. For the metabolite-transporting branch, the positions equivalent to SMCT1’s substrate binding site residues are highlighted in blue. d, Sliced view of SGLT1_conHA (left panel) and SMCT1-butyrate (right panel). e, The superimposed SGLT1 (inward-facing) and SiaT (outward-facing). Zoomed-in view of regions that undergo considerable conformational changes are shown in blue boxes on the right (unrelated helices or loops are removed for clarity). The shift of TM helices between SiaT (gray) and SGLT1 (orange) is indicated by black arrows. From outward-open to inward-facing conformation, the N-terminal half of TM10 undergoes significant inward movement around a Gly-Pro-Pro motif at the center of the helix. Concomitantly, the short loop connecting TM9-TM10 and the C-terminal part of TM9 also moves inward. As a result, the N-terminal end of TM10 and the TM9-TM10 loop come into contact with TM2 and EL4 of the extracellular domain, which collapses the extracellular vestibule and stabilizes the closed conformation of the extracellular gate. Phe453^TM10, at the end of TM10, thus moves into a position to contact other extracellular gate residues to shield the substrate-binding pocket from the extracellular solution. In association, TM11 and TM12 tilt away to accommodate the movement of TM10. On the intracellular side, TM5, together with TM4, tilts outward while TM8 and TM9 tilt away from TM1, TM5 and TM6. These movements open the intracellular entrance of the vestibule and widen the permeation pathway to enable substrate release. The increased distance between TM8 and TM1/TM5 is linked with the disruption of both Na2 and Na3 sites. Thus, Na⁺ binding is coupled to the conformational changes during state transitions and glucose transport. During state transitions, the extracellular lid domain is also expected to undergo significant conformational changes, which might help stabilize conformational states or give rise to distinct surface features to modulate Na⁺ transport. f, Structural comparison of SGLT1_conHA (orange), SMCT1 (cyan) and vSGLT (gray, PDB: 3DH4). g, Structural comparison of SGLT1_conHA (orange), SMCT1 (cyan). The orientation difference of the extracellular domain is zoomed-in in red box.