I am doing semi-rational design of my protein. So far I have one mutation that increases thermostability identified in vitro. However, Rosetta didn't predict this mutation in pmut scan. When I scored the protein variant with this mutation, the score was better than all the mutations predicted by Rosetta.
Now I'm trying to see if there is a combination of mutations (using predicted mutations and invitro predicted mutation) that will give me the best energy, but instead of the predicted mutations making the invitro predicted variant's score even better, they are actually lowering it by a lot.
Does anyone have an idea of what might be going on?
I'll appreciate any suggestions. Thanks!