Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. We describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic N-and C-termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We could therefore build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions. KeywordsAquaporin-0; lens; MIP; two-dimensional crystal; lipid-protein interaction; electron crystallography Members of the aquaporin (AQP) family form membrane pores that are either highly selective for water (aquaporins) or also permeable to other small neutral solutes such as glycerol and urea (aquaglyceroporins) (reviewed in 1 ). Structural studies have revealed that all AQPs share the same basic architecture, which consists of two tandem repeats, each containing a bundle of three transmembrane α-helices and a hydrophobic loop with the highly conserved asparagine-proline-alanine (NPA) motif 2 -8 . The two NPA-containing loops B and E fold back into the membrane and form short α-helices (HB and HE) that line the water pore. The ar/R constriction site, so named because it is formed by an aromatic and an arginine residue, confers water selectivity to AQP pores, while the NPA motifs play an important role in the proton exclusion mechanism (reviewed in 9 ).Correspondence to: Thomas Walz.Correspondence and requests for materials should be addressed to T.W. (twalz@hms.harvard.edu). Coordinates and structure factors for junctional and non-junctional AQP0 have been deposited in the Protein Data Bank (accession codes 2B6O and 2B6P, respectively).. Suplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. AQP0 is the most abundant protein in lens fibre cell membranes, where it forms not only water pores but also the 11-13 nm "thin lens junctions" that assemble upon proteolytic cleavage of the cytoplasmic termini 10 , 11 . We recently presented the structure of the AQP0-mediated membrane junction at 3 Å resolution as determined by electron crystallography of doublelayered two-dimensional (2D) crystals 7 . The structure showed that AQP0 junctions are stabilised by specific interactions between tetramers in adjoining membranes involving almost exclusively proline residues. Calculated pore profiles also showed that the pore in junctional AQP0 is highly constricted due to a substantially ...

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Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.connexin channels ͉ electron crystallography ͉ intercellular communication ͉ membrane protein structure ͉ two-dimensional crystals

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