I've been using the ddg_monomer application with high resolution protocol flags and the alternative mutation file format (to obtain more than one mutation) on the constant domain of a TCR (alpha and beta chain).
I'm interested in 3 ddg values:
- With a single mutation on the alpha chain (mut1)
- With a single mutation on the beta chain (mut2)
- With the pair of mutations (mut1+2)
However, in one set of mutation I'm introducing two cysteines (1 on the alpha chain + 1 on the beta chain) - to yield a disulfide bond between the two chains. (These mutations are based on the observation of this specific disulfide bond at the same positions in another specie…)
So I was expecting to obtain ddg values > 0 for mut1 and mut2 and for mut3 an increase in stability (lower ddg) due to the disulfide bond.
However, I obtain a higher ddg value for the mut1+2.
ddG: description total fa_atr fa_rep fa_sol fa_intra_rep fa_elec pro_close hbond_sr_bb hbond_lr_bb hbond_bb_sc hbond_sc dslf_fa13 coordinate_constraint an
ddG: T48C 1.089 1.728 0.654 -2.855 0.003 2.586 0.007 -0.026 -0.836 0.397 1.041 0.008 0.000 0.000
ddG: S147C 1.842 -1.355 1.629 -0.665 0.015 0.092 -0.007 -0.009 0.100 0.465 1.443 -0.002 0.000 0.000
ddG: T48CS147C 2.450 1.173 0.950 -5.977 0.006 6.373 0.012 0.042 -0.595 1.295 1.387 0.005 0.000 0.000
I see in the output pdb's that the disulfide bond is not made. Is there a way to help ddg_monomer along with the formation of the disulfide bond?
I then tried to force the disulfide bond (after the ddf_monomer analysis) using
the -in:fix_disulf with a txt-file containing the positions of the cysteines:
score_jd2.linuxgccrelease -database /services/tools/rosetta/2016.20/main/database -in:file:silent mut_T48CS147C.out -in:file:fullatom -out:pdb -overwrite -score:weights talaris2014 -out:file:scorefile score_dis.sc -in:fix_disulf dis.txt
Followed by minimization of the structure with the forced disulfide bond:
relax.default.linuxgccrelease -in:file:s mut_T48CS147C_round_1_0001.pdb -score:weight talaris2014 -in:fix_disulf dis.txt
And then the score was calculated again:
score_jd2.linuxgccrelease -database /services/tools/rosetta/2016.20/main/database -in:file:s mut_T48CS147C_round_1_0001_0001.pdb -in:file:fullatom -out:pdb -overwrite -score:weights talaris2014 -out:file:scorefile score_cc_single.sc
This seemed to "fix" the problem - I now have a disulfide bond. But then I obtained a non-comparable "total score"-value, as this was relaxed. So therefore I relaxed the pdb file, with the mut1+2 without the forced disulfide bond, and obtained an ever lower "total score". Thus I once again ended up with the problem that the introduction of the disulfide bond was not favorable (as I expected it to be).
Are there anything you can suggest to solve this ddg_monomer+disulfide problem for me?