Calcin sequence alignment and evolutionary analysis. (A) The eight calcins known to date are aligned with opicalcin₁ as reference by Clustal Omega. The identity values to opicalcin₁ are shown on the right side. Positively charged residues lysine (K) and arginine (R), negatively charged residues aspartic acid (D) and glutamic acid (E), and the disulfide bond-forming cysteine (C) are highlighted by the colors blue, red, and gray, respectively. Columns with identical residue are marked by asterisks, whereas columns with only one difference are marked by colons on the bottom. (B) Three pairs of highly conserved disulfide bonds (Cys³-Cys¹⁷, Cys¹⁰-Cys²¹, and Cys¹⁶-Cys³²) and the residues forming part of β strands appear connected to form an ICK motif. (C) The evolutionary tree is built with the principle minimum evolution by MEGA 5.2. The genetic distance is measured by Poisson correction method with the formula d_AB = −ln(1 − f_AB), where f_AB is the fraction of different amino acids after the Poisson distribution between two sequences (dissimilarity).