Diagram of alpha-helix-based evaluation. (A) Stacked positions in alpha helices have been observed to coevolve to maintain ionic interactions which stabilize the helix. The periodicity of the alpha helix is 3.6 residues per turn, so stacked positions are the (i, i + 3) and (i, i + 4) position pairs, where i is the position number. K133/E136 form an (i, i + 3) pair capable of an ionic interaction; D141/K145 are an (i, i + 4) pair capable of an ionic interaction. The structure is a single helix extracted from 1 MBD, a sperm whale myoglobin structure, diagramed with PyMol. (B) The distribution of coevolution scores from (i, i + n) position pairs are compared against a background distribution generated from all other position pairs in the same alignment using a one-tailed, two-sample t-test. The scale of the y-axes differ due to the large difference in the number of position pairs in each distribution. The p-values resulting from these tests are those presented in this document. These t-test p-values were compared against p-values generated with other statistical tests to confirm the applicability of the t-test to these data (see Methods).