3.

Ca²⁺ binding in the NaK selectivity filter. (a) 2F_o-F_c ion omit map (1.5σ) of the NaKNΔ19-Na⁺ complex co-crystallized with 10mM CaCl₂, showing electron density for a Ca²⁺ ion (purple sphere) at the external entrance. Na⁺ and the contaminant at site 3 are modeled as green and orange spheres, respectively. Electron density for the protein backbone is also shown as a blue mesh. (b) F_{0.5mM Ca} − F_{0mM Ca} (upper) and F_{5mM Ca²⁺} − F_{0mM Ca²⁺} (lower) difference map contoured at 10 σ showing increasing intensity of Ca²⁺ binding upon increasing Ca²⁺ concentration. Dotted lines indicate the position of central axis along which the 1-D electron density was calculated. (c) 1-D electron density profile of Ca²⁺ binding at the external entrance showing increased Ca²⁺ binding at increasing concentrations. The control is from a difference map between two native crystals grown in the absence of Ca²⁺. (d) Occupancy of Ca²⁺ binding at the external site was calculated by integration of the peaks in 1-D electron density profile and normalizing them against the 10 mM Ca²⁺ peak. The plot of occupancy against Ca²⁺ concentration give rise to a K_d value of about 1.8 mM upon fitting the curve to a single component Langmuir equation.