Overview of LacY. The structure shown corresponds to that of the inward closed conformation as obtained from our equilibrium MD simulations with Glu-269 and Glu-325 protonated and deprotonated, respectively (19). Before our SMD simulations, this structure was subjected to additional equilibration (see text). N- and C-domains are shown in brown and green, respectively. Residues 30–40 and 136–166 (N-domain; helix I and V, respectively) and 253–287 (C-domain; helix VIII) are presented as ribbons. Sugar in the centrally located, water-rich binding cavity is shown (without hydrogen atoms) in gray and red along with the side chain of Phe-27. The lumen of LacY is defined to start at the Phe-334/Tyr-350 constriction and end at the Ile-40/Asn-245 constriction. Water molecules at the vestibules, i.e., above Phe-334/Tyr-350 or below Ile-40/Asn-245, as well as within the lumen, are shown in red and white. Regions I (cytoplasmic half-channel) and II (periplasmic half-channel) are indicated by double arrows in red and blue, respectively.