I have a problem in 2D PMF calculations.
There are two reaction coordinates (RC) in my system. RC1 stands for conformational change of protein (saying RC1 ranges > from 1 to 10, where RC1=1 means closed state of protein, RC1=10 means open state), and RC2 is the distance between ligand and active site (RC2 ranges > from 1 to 10, where RC2=1 means ligand bound state, RC2=10 means ligand escaped state). After the time-consuming umbrella sampling calculations (with amber9) on two RCs, the 2D free energy profile can be obtained with WHAM program. However, the open state of protein with ligand bound to active site (RC1=10, RC2=1) is found to be a minimum on the free energy surface, while the closed state with ligand bound has higher energy (RC1=1, RC2=1). This is opposite to what I have expected, where the closed state of protein with ligand bound should be a stable state.
Then the umbrella sampling data of different RC1 (from 1 to 10) with fixed RC2 (RC2=1) are used to get quasi-1D free energy profile of protein conformational change with ligand bound, and the results seem to be reasonable: the open state is energetic unfavorable, and the closed state has a minimum. The system stability in quasi-1D free energy profile is reasonable.
Besides, umbrella sampling on RC1 (no bias potential is imposed on RC2, and the ligand position and orientation is almost unchanged because there are lots of favorable interactions between ligand and active site is strong) was done on the same system. The generated 1D-PMF results is quite similar to the quasi-1D one, and quite different to the 2D-PMF results when RC2=1.
All the structures as well as the data have been carefully checked. Everything looks good, but the results look strange. I do not understand why the stability of the system in 2D-PMF and quasi-1D-PMF is different. As the results from 2D-PMF are inconsistent with common knowledge of protein-ligand complex, I think there may be something wrong with my 2D-PMF calculations. Can anyone give some suggestions? Thanks in advance!