Figure 3.

Rosetta modeling-guided double and combinatorial mutagenesis of 9°N-N. (A) Classification of variants based on the site of amino acid substitution and combinatorial status. Substitutions leading to an increase in NP-DNA polymerase activity are highlighted in green, the variant with the highest catalytic rate is highlighted in blue. (B–G) Rosetta-based structural models of selected key substitutions, highlighting the second-shell effects on the catalytic site (B,C) and the distal finger subdomain (D–G). Active site metal ion Mn²⁺ is in purple. Residues are drawn as sticks with color by atom (carbon, white; nitrogen, blue; oxygen, red; phosphorus, orange). Dashed yellow line indicates the second shell interaction.