Three-Dimensional Fold of Human AQP1 Water Channel Determined by Electron Cryo-Crystallography of 2-Dimensional Crystals Embedded in Ice

Abstract

The entry and exit of water across the cell membrane is a fundamental physiological process. The observed high permeability of water in the case of mammalian erythrocytes and renal proximal tubules led to the discovery of water channels (aquaporins, AQP) (Denker et al., 1988; Smith and Agre, 1991). Aquaporins are members of the MIP (major intrinsic protein) superfamily (Gorin et al., 1984) and are found in both eukaryotes and prokaryotes where they serve as channels for rapid dissipation of osmotic gradients across the lipid bilayer (Agre et al., 1995; Verkman et al., 1996). In order to understand the structural elements that constitute and control the water-selective permeability of AQP1, a detailed knowledge of the high-resolution 3-D structure in the bilayer is essential. To this end, 2-D crystals of AQP1 have been used to examine the structure using electron crystallography. Projection maps of AQP1 were determined by electron cryo-crystallography by three groups (Jap and Li, 1995; Mitra et al., 1995; Walz et al., 1995) who used different conditions for 2-D crystallization and specimen preservation for microscopy. Subsequently, a 7Å resolution, 3-D density map of AQP1 was determined by us (Cheng et al., 1997) from an analysis of minimal dose images and electron-diffraction patterns recorded from ice-embedded from ice-embedded 2-D crystals. 3-D density maps, at a similar resolution were reported by the other 2 groups (Li et aL9 1997; Walz et al., 1997) which were determined using specimens preserved in trehalose and the tilted projections comprised of images and electron diffraction patterns (Walz et al., 1997) and only images (Li et al.9 1997).