A) Upper: Overview of the AB tetramer, with each of the monomers depicted in a different colour. The front monomer has been removed for clarity. The black box indicates the region expanded in the lower panel. Lower: An expanded view of the selectivity filter, showing only two monomers as pink and white ribbon cartoons with the selectivity filter residues drawn as sticks in the corresponding colours. Sodium ions are shown as cyan spheres and well-ordered waters as red spheres (at 0.4x their actual radii, for clarity). The final refined 2Fo-Fc electron density maps for the protein, ions and water are shown at 2.0 σ in dark grey mesh and 0.8 σ in light grey , and the initial difference map (without sodiums or waters included in the phase calculation) at 3.0 σ in green mesh.B) Superposition of the sodium ion binding sites found in the crystallographically-distinct AB and CD tetramers and the proposed binding sites from other studies. The tetramers were superposed with the LSQKAB program (Kabsch, 2010) from CCP4 (Winn et al., 2011) using residues 131-220 from all four monomers in each tetramer. Only two monomers from the AB tetramer are shown for clarity, and coloured as in panel A. Sodium ions from the AB tetramer are depicted as cyan spheres, those from the CD tetramer as teal spheres. Predicted sodium ion binding sites from Ulmschneider et al. (2013) are indicated as small black spheres and labelled on the right of the spheres according to the nomenclature (S0 to S4) in that study. Predicted sodium ion binding sites from Payendeh et al. (2011), based on a closed channel structure of the close orthologue NavAb, are indicated on the far right and labelled as per the nomenclature of that study (SiteHFS, SiteCEN and SiteIN). This and subsequent molecular figures were drawn with PyMol (The PyMOL Molecular Graphics System, Version 1.6, Schrödinger, LLC) unless otherwise stated. 