Interestingly, within the conformer-specific regions of differing dissociation is a known calcium-binding motif between residues 93-100. The extended conformer formed c (1), y (4) and z (5) fragments in this region, compared with only one z ion in the compact, suggesting that this site could be used for calcium binding within the compact structure but not the more extended form. This hypothesis fits with the PDB structure, literature, and the intact mass measurement (Supplementary Figure ), which suggests the majority of calmodulin has 2-3 calcium occupancy in this state, compared to 3-4 for the compact conformer. Three other known calcium-binding sites (colored cyan in Figure ) showed similar fragmentation between the two structures, consistent with comparable calcium occupation and their locations away from the solvent-accessible central helix. These sites did show slightly reduced fragmentation compared to surrounding regions, relative to their proximity to the protein termini where high levels of bond dissociation are expected, supporting the idea that within these regions the backbone would be protected from ECD fragmentation by calcium binding.