Structure of the Water Channel AqpZ from Escherichia coli Revealed by Electron Crystallography P. Ringler 1 , M. J. Borgnia 2 , H. Stahlberg 1 , P. C. Maloney 3 , P. Agre 2 and A. Engel 1 * 1 M.E. Mu È ller Institute for Microscopy, Biozentrum University of Basel Klingelbergstrasse 70 CH-4056, Basel, Switzerland 2 Department of Biological Chemistry, Johns Hopkins University School of Medicine Baltimore, MD 21205, USA 3 Department of Physiology Johns Hopkins University School of Medicine, Baltimore MD 21205, USA Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and speci®c diffusion of water molecules. Aquaporins are present in animals, plants, algae, fungi and bacteria. Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli. A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and puri®ed to homogeneity. Solu- bilized with octylglucoside, the puri®ed AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetra- gonal crystals with unit cell dimensions a  b  95 A Ê , g  90  when reconstituted by dialysis in the presence of lipids. Three-dimensional reconstruction of negatively stained lattices revealed the p42 1 2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1). The 8 A Ê projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins. # 1999 Academic Press Keywords: aquaporin; major integral protein family; 2D crystallization; electron crystallography; STEM *Corresponding author Introduction As solvent for the chemical processes of life water passes across membranes by diffusion either through the bilayer or through highly speci®c water pores, the aquaporins. Their presence has been postulated in special membranes exhibiting high osmotic permeability, because the activation energy for the diffusion through the lipid is high (reviewed by Finkelstein, 1987). The discovery of the ®rst aquaporin (AQP1) in the human red cell membrane provided the molecular explanation for selective osmotic water ¯ow across cell membranes (Preston & Agre, 1991). Aquaporins with multiple physiological functions have been reported in mammals and have been shown to be involved in several clinical disorders (for a review, see King & Agre, 1996). Mammalian aquaporin mutants have been identi®ed with phenotypes including lens cat- aract (MIP, the major intrinsic protein of lens ®ber cells; Shiels & Bassnett, 1996), loss of the red cell Colton blood group antigens (AQP1; Agre et al., 1995), nephrogenic diabetes insipidus (AQP2, the water channel of the collecting duct; Deen et al., 1995), and incomplete renal ¯uid concentration (AQP4; Verkman et al., 1995). Aquaporins have also been found in frogs (Abrami et al., 1994), insects (Le Cahe Ârec et al., 1996), plants (Weig et al., 1997) and bacteria (Calamita et al., 1995). This widespread occurrence in plant and animal king- doms suggests an essential physiological function. Discovery of the aqpZ gene in the wild-type Escherichia coli was achieved by homology cloning using the sequence-related bacterial gene of the glycerol facilitator (glpF). The aqpZ DNA from E. coli contains a 693 base-pair open reading frame encoding a polypeptide whose sequence is 28-38 % identical to those of other known aquaporins (Calamita et al., 1995). Comparative transport analysis using injection of AqpZ cRNA in Xenopus oocytes and assays for water or glycerol per- meability demonstrated the high water-selectivity E-mail address of the corresponding author: aengel@ubaclu.unibas.ch Abbreviations used: AqpZ, aquaporin Z; AQP1, aquaporin 1; 2D, two-dimensional; 3D, three- dimensional; DMPC, 1,2-dimyristoyl-sn-glycero-3- phosphocholine; IQ value, measure of the signal-to- noise ratio in diffraction spots; LPR, lipid-to-protein ratios; MIP, major intrinsic protein; MSA, multivariate statistical analysis; OG, octyl-b-D-glucopyranoside; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Article No. jmbi.1999.3031 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 291, 1181±1190 0022-2836/99/351181±10 $30.00/0 # 1999 Academic Press