# RBFE for 8 simultaneous mutations **URL:** \ **Category:** pmx\ **Created:** [June 15, 2026, 6:25am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169 "2026-06-15T06:25:48Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![Sankalp](https://avatars.discourse-cdn.com/v4/letter/s/aca169/32.png) [@Sankalp](https://ask.bioexcel.eu/u/Sankalp)\ **Post date:** [June 15, 2026, 6:25am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/1 "2026-06-15T06:25:48Z")
Hii, I have a beta-sheet protein of 128 residues, of which 8 are tyrosines. I have mutated all 8 TYR simultaneously to PHE. I used the same mdp files, except the temperature was changed to 300K. I followed the PMX protein mutation tutorial, created 50 windows for forward and reverse, and performed the final 80 ps equilibration. I got the result as shown. The number seems too high after I divide it by 8. What could be the issue? Should I increase the equilibration run length, or perform mutations sequentially rather than simultaneously? ![wplot](https://europe1.discourse-cdn.com/flex013/uploads/bioexcel/original/2X/7/72aff1de95eb28ead6bab5eec6f61d87138a40a4.png) ---
**Author:** ![bgroot](https://avatars.discourse-cdn.com/v4/letter/b/dc4da7/32.png) [@bgroot](https://ask.bioexcel.eu/u/bgroot)\ **Post date:** [June 15, 2026, 8:16am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/2 "2026-06-15T08:16:40Z")
Hi, you don’t mention if you subtracted a reference case (such as the TRP-\>PHE mutation in the unfolded state, for which you can use a tripeptide as an approximation; in this case you should apply this correction 8 times). Without that, the raw free energy numbers are meaningless. Also, dividing by 8 might not be so informative, as it will only give you the average effect per mutation, but likely some of the mutations contribute a lot more than others, depending on the position. ---
**Author:** ![Sankalp](https://avatars.discourse-cdn.com/v4/letter/s/aca169/32.png) [@Sankalp](https://ask.bioexcel.eu/u/Sankalp)\ **Post date:** [June 17, 2026, 4:19am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/3 "2026-06-17T04:19:35Z")
Hi, thank you for the response. The purpose of this calculation is to calculate the effect of mutation on protein dimerization. As TYR residues are a dimerization site, I am mutating them. I am using the thermodynamic cycle shown in the attached figure. till now I simulated only the lower leg, where I mutated TYR of monomer to PHE of monomer, so this value of ~568kJ/mol corresponds to ΔGmonomermutation, which I think is very high. What correction factor should I use here? ![sketch1781669493692](https://europe1.discourse-cdn.com/flex013/uploads/bioexcel/original/2X/2/23e40b0acddfb7bf12d466493aefe58a6e743f67.jpeg) ---
**Author:** ![bgroot](https://avatars.discourse-cdn.com/v4/letter/b/dc4da7/32.png) [@bgroot](https://ask.bioexcel.eu/u/bgroot)\ **Post date:** [June 17, 2026, 8:39am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/4 "2026-06-17T08:39:17Z")
the thermodynamic cycle looks fine. The number you get only makes sense relative to the upper leg (dimer). So if you subtract the monomer value from the dimer value, you know how much the mutations affect the dimerization. The individual monomer and dimer mutation dG values have no physical meaning. ---
**Author:** ![Sankalp](https://avatars.discourse-cdn.com/v4/letter/s/aca169/32.png) [@Sankalp](https://ask.bioexcel.eu/u/Sankalp)\ **Post date:** [June 18, 2026, 12:41pm UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/5 "2026-06-18T12:41:32Z")
Thanks for the response. I ran the dimer leg and calculated the final values for monomer Bootstrap (Std Err): iteration 100/100 Bootstrap (Conv): iteration 100/100 BAR: dG = 568.22 kJ/mol BAR: Std Err (analytical) = 1.56 kJ/mol BAR: Std Err (bootstrap) = 1.25 kJ/mol BAR: Conv = 0.25 BAR: Conv Std Err (bootstrap) = 0.15 for dimer Running Nelder-Mead Simplex algorithm… Bootstrap (Std Err): iteration 100/100 Bootstrap (Conv): iteration 100/100 BAR: dG = 581.80 kJ/mol 🛑 **BAR: Std Err (analytical) = 10.20 kJ/mol**🛑 BAR: Std Err (bootstrap) = 1.69 kJ/mol BAR: Conv = 0.97 BAR: Conv Std Err (bootstrap) = 0.00 The gap between forward and backward leg for dimer is quite high, and distributions are not overlapping, and I think that’s why the **BAR: Std Err (analytical) = 10.20 kJ/mol** is coming high. Should I worry about this? As the free energy value seems to be in the range, but I don’t have experimental validation. ![image](https://europe1.discourse-cdn.com/flex013/uploads/bioexcel/original/2X/d/d9e2d431153594b11bbfa6962ef48f4188a21794.png) ---
**Author:** ![bgroot](https://avatars.discourse-cdn.com/v4/letter/b/dc4da7/32.png) [@bgroot](https://ask.bioexcel.eu/u/bgroot)\ **Post date:** [June 19, 2026, 7:36am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/6 "2026-06-19T07:36:38Z")
the lack of overlap is likely because you’re doing 8 mutations at once, which is a big perturbation. This can be remedied by making slower (2 fold or 5 fold) transitions. ---
**Author:** ![Sankalp](https://avatars.discourse-cdn.com/v4/letter/s/aca169/32.png) [@Sankalp](https://ask.bioexcel.eu/u/Sankalp)\ **Post date:** [June 22, 2026, 7:28am UTC](https://ask.bioexcel.eu/t/rbfe-for-8-simultaneous-mutations/6169/7 "2026-06-22T07:28:58Z")
Thank you for the help. Now I used 500 ps equilibration per frame. And I am getting a low error bar. Thanks again! Running Nelder-Mead Simplex algorithm… Bootstrap (Std Err): iteration 100/100 Bootstrap (Conv): iteration 100/100 BAR: dG = 578.12 kJ/mol BAR: Std Err (analytical) = 1.01 kJ/mol BAR: Std Err (bootstrap) = 0.89 kJ/mol BAR: Conv = 0.00 BAR: Conv Std Err (bootstrap) = 0.13 I ![wplot](https://europe1.discourse-cdn.com/flex013/uploads/bioexcel/original/2X/1/113b1532dd95016ec698b1c596a220579aaf69d5.png)