The 3.7 angstrom projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypa l member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubili zed GlpF protein revealed a tetrameric structure of similar to 80 Angstrom side length. Scanning transmission electron microscopy yielded a mass of 17 0 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 Angstrom resolution. Cryoelectron micros copy provided a 3.7 Angstrom projection map exhibiting a unit cell comprise d of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is d istinctly larger in GlpF than in AQP1.