Three-dimensional fold of the human AQP1 water channel determined at 4 angstrom resolution by electron crystallography of two-dimensional crystals embedded in ice

Here, we present a three-dimensional (3D) density map of deglycosylated, hu man erythrocyte aquaporin 1 (AQP1) determined at 4 Angstrom resolution in p lane and similar to 7 Angstrom resolution perpendicular to the bilayer. The map was calculated by analyzing images and electron diffraction patterns r ecorded from tilted (up to 60 degrees), ice-embedded, frozen-hydrated 2D cr ystals of AQP1 in lipid bilayer membranes. This map significantly extends t he findings related to the folding of the AQP1 polypeptide chain determined by us at a lower, 7 Angstrom by similar to 20 Angstrom, resolution. The so lvent-accessible volume within a monomer has a vestibular architecture, wit h a narrow, similar to 6.5 Angstrom diameter constriction near the center o f the bilayer, where the location of the water-selective channel is postula ted to exist. The clearly resolved densities for the transmembrane helices display the protrusions expected for bulky side-chains. The density in the interior of the helix barrel (putative NPA box region) is better resolved c ompared to our previous map, suggesting clearer linkage to some of the heli ces, and it may harbor short stretches of alpha-helix. At the bilayer extre mities, densities for some of the inter-helix hydrophilic loops are visible . Consistent with these observed inter-helix connections, possible models f or the threading of the AQP1 polypeptide chain are presented. A preferred m odel is deduced that agrees with the putative locations of a group of aroma tic residues in the amino acid sequence and in the 3D density map. (C) 2000 Academic Press.