(A) Ribbons diagram of the DhaPylSc homodimer colored by subunit and secondary structure. Subunit A is colored with α-helix in red, β-sheet in yellow, and random coil in green. Subunit B is colored with α-helix in cyan, β-sheet in magenta, and random coil in pink. (B) Structure-based sequence alignment of DhaPylSc and MmzPylS C-terminal domain. The amino acids are highlighted by secondary structure (same color scheme as Subunit B in Fig. 1A) and critical residues associated with binding pyrrolysine are colored in green. (C) Structural overlap of the B-subunit of DhaPylSc colored in red and MmzPylS C-terminal domain colored in cyan. The novel regions in the DhaPylSc structure are colored in slate. (D) Pyrrolysine binding pocket in DhaPylSc. Adenylated pyrrolysine analog colored yellow is modeled into the binding pocket according to its structurally equivalent position in MmzPylS. Trp139 residue shown in stick leads to a much tighter pyrrolysine pocket. (E) Pyrrolysine binding pocket in MmzPylS. Leu309 in structurally equivalent position as Trp139 in DhaPylSc is shown as stick.