Gap junction channels connect the cytoplasms of apposed cells via an intercellular conduit formed by the end-to-end docking of two hexameric hemichannels called connexons. We used electron cryomicroscopy to derive a three-dimensional density map at 5.7 Å in-plane and 19.8 Å vertical resolution, allowing us to identify the positions and tilt angles for the 24 α helices within each hemichannel. The four hydrophobic segments in connexin sequences were assigned to the α helices in the map based on biochemical and phylogenetic data. Analyses of evolutionary conservation and compensatory mutations in connexin evolution identified the packing interfaces between the helices. The final model, which specifies the coordinates of C α atoms in the transmembrane domain, provides a structural basis for understanding the different physiological effects of almost 30 mutations and polymorphisms in terms of structural deformations at the interfaces between helices, revealing an intimate connection between molecular structure and disease.