Steric clash of CAM with the hexameric Orai pore. PYMOL-generated cartoon representations of the hexameric Drosophila Orai crystal structure are shown with and without CaM C lobe. (A) View of dOrai from the cytosolic face. The aligned human CBD_Orai1 structure (with W76 side chain) is shown in yellow. Glutamate side chains near the extracellular end of the pore are shown for orientation. Helices are color coded: TM1 (red), TM2 (teal), TM3 (blue-gray), and TM4 (blue). (B) dOrai with a surface representation of the CaM C lobe (green) bound to CBD_Orai1. (C) Side view of the dOrai TM1 helices with Ca²⁺–CaM bound to the aligned human CBD_Orai1 structure (yellow) highlights the steric clash of the C lobe with the adjacent Orai pore helices. Overlap extends in the vertical direction to R83. (D) Cutaway cytosolic view of the dOrai TM1 helices in complex with Ca²⁺–CaM shows that the clockwise adjacent TM1 helix (arrow) overlaps completely with CaM. (E) Cutaway cytosolic view of the dOrai–CaM C lobe complex after a 15-Å radial dilation of each TM1 helix (red, compare with B), necessary to remove overlap between the adjacent TM1. Note that CaM now clashes with the TM4 cytosolic domain (blue) and overlaps completely with TM3 (blue-gray). (F) The TM1 pore regions for two opposite dOrai subunits are shown together with the pore-lining side chains (corresponding human Orai1 residues are indicated in parentheses).