Structure-guided combinatorial library design workflow. For clarity, only one TadA*8.20 monomer (green) with ssDNA (yellow) is shown. The 23 substitution sites identified in the first round of evolution are shown as violet spheres in the first panel. Based on the structures of TADAC-1 variants (Fig. 2), 10 sites were selected, 8 from TADAC-1 (violet) plus 2 rationally selected (cyan), in the loop between α1 and β1, α2-helix, active site, the loop between β4 and β5 and α5-helix, to generate the TADAC-3 variants (second panel). The substitution sites in TADAC-2 from the second round of evolution are shown as violet (same sites identified in TADAC-1) and orange (new sites) spheres in the TadA*8.20 structure (third panel). To generate the TADC-1 variants, eight substitutions from the second round of evolution were added to TACAC-3.154 (fourth panel).
