Double system/single box simulation - Free energy calculation

Dear all,

I want to calculate the relative binding free energy difference of a complex, with alchemical transformation using the double system/single box approach. Is there any suggested tutorial about this approach? I followed the tutorial on the pmx page (Mutation free energy calculations) but this covers a mutation in a single protein complex.

I am interested to see the free energy difference of an arginine to alanine (R2A) mutation. Based on the previous tutorial and the Patel et al. (2021) paper ( I did the following:

  • Hybrid topology generation with the pmx server. Then, I build the boxes manually containing the complex with the wild-type protein-ligand and the mutated ligand, and vice-versa.
  • 100 ns equilibrium run. Then I discarded the first 20 ns and selected 100 snapshots from the rest of the run for the transition simulations.
  • For each snapshot, 500 ps forward and backward runs were performed changing the lambda from 0 → 1 and from 1 → 0 respectively.
  • Finally I calculated the DeltaG with pmx analyse using the dhdl.xvg files for each snapshot run.

The value I got is 1176.32 ± 5.32 kJ/mol (281.15 ± 1.27 kcal/mol) which is much greater than expected. I am repeating the transition runs using 10 ns.

Should I take something else into account? Any suggestion will be very welcomed.