The parameters for the incoming nucleotide were previously reported . Protonation states for ionizable residues were calculated with ProPKA, and hydrogens were added based on the protonation states of the previous step, as well as clashes were checked and resolved using MolProbity . The LeAP 52 module of AMBER18 were used to neutralize and solvate the system. The ff14SB , OL15 and TIP3P forcefields were used for protein, DNA, and water (along with ions) respectively. Based on the volume of both the systems, 73 Mg 2+ and Mn 2+ ions and 146 Cl -ions were added to the systems to make 20mM solutions of MgCl2 and MnCl2 respectively. The final atom count was 308356 atoms for the system with Mg 2+ ions and 310729 atoms for the Mn 2+ system.