Similarity plot and alignment of mid domain sequences. (A) A two-dimensional cluster graph groups mid domain sequences. Nodes represent sequences connected by grayscale edges (high to low similarity colored black to gray). Three groups include Ago sequences (magenta), piwi-like sequences (green), and nematode paralogs (cyan). Sparsely connected nodes are colored by superkingdom: eukaryotes (black), bacteria (blue), and archaea (red). Representatives are labeled with species abbreviation and protein name or PDB id. Species abbreviations: Aa, A. aeolicus; Af, A. fulgidus; At, A. thaliania; Ce, C. elegans; Dm, D. melanogaster; Hs, H. sapiens; and Sp, S. pombe. (B) Ago/piwi multiple sequence alignment highlights conservations. Sequences are labeled and colored as in (A). Residue positions are colored by Ago conservation: hydrophobic (yellow), small (grey), and conserved polar (black). Residue numbers are to the left of the alignment, with omitted residues in brackets. Observed secondary structures (E strand and H helix) are above the alignment, while predicted secondary structures are below. Ago2 MC is boxed, with identified aromatics highlighted magenta, locally similar secondary structures (to eIF4E) highlighted pink, and dissimilar regions (to eIF4E) highlighted light blue.