I have been trying to run a theoretical alanine scan on a protein.
Unfortunately, I cannot repeat the results I obtain when I run exactly the same experiment (just copy all files to a new directory and run again) more than one time. Attached is a png of the correlation between run 1 and 2. The black line is 1:1 correlation. You can see that some mutants look ok (green ring) while others are very badly different (red rings).
I have checked the structures (I output all 50 for the iterations) and see that there is almost zero difference between the structures in the region surrounding the mutation, and very minor differences in the loop regions. I’ve also attached a couple of images to demonstrate this. They are of the N47A (ddg is +3, or -6 REU in the repeat) mutation highlighted on the plot. The differences in the two TRP residues in the region of the mutant (pink sticks in the image) occur between the repacked WT and the mutant (but there is no discernable difference in packing between the mutant and the repeated mutant).
I have used the minimization prep as instructed, and included cst file for the calculation.
I have tried also increasing the number of iterations to 50 (correlation between repeats was even worse when only using 20 iterations) and also selecting the ddg::mean true flag, to see if this could even things out (Thanks to Julia K for the suggestions!) but still this is the best I can come up with.
Does anyone have any further suggestions? I really don’t mind if the absolute numbers are absolutely correct, I’m just looking to bucket into “stabilizing in presence of ligand” /”stabilizing in absence of ligand” / “stabilizing in both” / “destabilizing”, and would like to be able to reproduce a trend.