Figure 2: Crystal structures of Ca²⁺-bound and Ca²⁺-free Cx26 gap junction channels.

(a) Packing of Cx26 dodecamers (cyan) in the H32 lattice. The main crystal contact involves residues on the cytoplasmic surface of the channel. The two subunits in the asymmetric unit, a dimer of Cx26, are shown in green and orange. (b) Three-dimensional cryoEM map at 5.7 Å resolution in X, Y and 19.8 Å resolution in Z derived from electron cryocrystallography of two-dimensional crystals of human Cx43 channels in gap junction plaques⁵. (c) Ca²⁺-bound Cx26 structure (cyan) and the 2F_o−F_c electron density map (blue mesh) at 3.3 Å resolution and contoured at 1.5-σ. (d) Side view of the dodecameric assembly of Cx26 forming the GJC. Ca²⁺ atoms are shown as yellow spheres. Distances are measured between Cα atoms. (e) A cross-sectional view of the Ca²⁺-bound structure (cyan) and map (blue mesh) showing the funnel-shaped cytoplasmic region of the pore that narrows in proceeding to the extracellular vestibule. (f) Inter-subunit residues that form the Ca²⁺-binding sites, as revealed by the 3.3 Å resolution crystal structure. The difference density peaks (the F_o−F_c maps) indicate the unrefined positions of Ca²⁺ ions (green). Three binding sites are shown in this cytoplasmic view. (g) A close-up of one of the Ca²⁺-binding sites shown in f. (h) A cross-sectional view of the Ca²⁺-free structure (orange), shown with a 3.8 Å resolution 2F_o–F_c map (blue mesh). (i) A view of the Ca²⁺-free structure as shown in f for the Ca²⁺-bound structure, showing both 2F_o−F_c and F_o−F_c maps. Note the absence of any green difference density peaks in the F_o–F_c map. (j) A close-up of E47 in subunit A for the Ca²⁺-free structure. To better visualize the conformational change of E47, the molecule has been rotated significantly compared with the perspectives shown in g and i. Side chains are shown as sticks, with atoms coloured as: carbon, cyan (Ca²⁺ bound) or orange (Ca²⁺ free); oxygen, red; nitrogen, blue. The 2F_o−F_c maps (blue mesh) and the F_o–F_c maps (green) are contoured at 1.5-σ and 4-σ, respectively.