Our long-range goal is to obtain crystal structures of different conformations of the lactose permease of Escherichia coli (LacY) in order to understand the mechanism of lactose/H+ symport at the atomic level. LacY is a paradigm for the Major Facilitator Superfamily, as well as membrane proteins in general. Our first X-ray crystal structure of a conformationally restricted mutant of LacY (C154G) represents a major breakthrough as the first structure of a cation-coupled symporter. In the past grant period, we accomplished another breakthrough by solving an x-ray structure of wild-type LacY to a resolution of 3.6 E, an accomplishment that took well over a decade and required development of a new, general approach-maintaining bound phospholipids. By this means, we also improved resolution of the C154G LacY structure to a resolution of ~2.9 E and showed that sugar binding is an induced-fit phenomenon. However, all structures display the same inward-facing conformation: pseudo-symmetrical N- and C- terminal 6 transmembrane 1-helix bundles, most of which are irregular, surrounding a large internal hydrophilic cavity open to the cytoplasmic side and tightly closed on the periplasmic side. The residues that play major roles in galactopyranoside recognition and H+ translocation are clustered near the apex of the cavity and inaccessible from the periplasmic side. A mechanism consistent with the structure and many biochemical/biophysical approaches is proposed, the heart of which is alternative accessibility of the sugar- and H+-binding sites to either side of the membrane. Despite a wealth of biochemical/biophysical data showing that transport involves opening and closing of inward- and outward-facing cavities, structures are needed in a different conformation(s) in order to obtain the mechanism at the atomic level. We have obtained diffracting crystals of likely candidates that are approaching a resolution suitable for atomic model building. The main aims of this proposal are (i) to obtain structures of conformations of LacY other than inward facing; (ii) to obtain a structure of LacY that diffracts to a resolution sufficient to visualize bound water, which may play a direct role in H+ translocation. We will combine mutagenesis and chemical modification to induce conformations different from the inward-facing conformation, which is favored by crystallization. The proposed structures will be invaluable for understanding the mechanism of cation-coupled membrane transporters, a class of proteins that plays essential roles in many cellular functions and has broad impact on biology and medicine.