Figure 2.

Calcium-titration experiments by X-ray crystallography. (A) Calcium coordination at the Ca1, 3, 4, 5, and 6 sites. Electron density for calciums 1, 3, 4, 5, and 6 were generated from F_o–F_c omit maps from structures soaked in 0, 50, 100, 250, 500, 1000, 5000, and 10 000 μM Ca²⁺, corresponding to PDB IDs: 4N20, 4N22, 4N24, 4N25, 4N26, 4N28, 4N2A, and 4N2B, respectively. Density was contoured at the 8σ level from F_o–F_c omit maps where calcium was removed and simulated annealing was performed during refinement. The electron density of each calcium was normalized to a water molecule that was present in all structures and whose density was not affected by soaking with calcium. (B) Calcium dissociation constants (K_D(app)) were determined by calculating the percent occupancy of calcium from the e/ų levels for each calcium at eight different soak concentrations, and two structures were solved at each soak concentration for data acquisition (n = 2 for each data point). (C) Sequence alignment of the PADs shows the conserved calcium binding residues (*) in the calcium switch. This region is highly conserved (red) among the PADs except for PAD6, which shows no enzymatic activity.