# Pmx RBFE gives oddly large deltaG **URL:** \ **Category:** pmx\ **Created:** [August 12, 2022, 4:46pm UTC](https://ask.bioexcel.eu/t/pmx-rbfe-gives-oddly-large-deltag/3847 "2022-08-12T16:46:56Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![k-zator](https://avatars.discourse-cdn.com/v4/letter/k/5daacb/32.png) [@k-zator](https://ask.bioexcel.eu/u/k-zator)\ **Post date:** [August 12, 2022, 4:46pm UTC](https://ask.bioexcel.eu/t/pmx-rbfe-gives-oddly-large-deltag/3847/1 "2022-08-12T16:46:56Z")
Hello, I’m new to pmx still and have been trying to run RBFE for a macrocycle host and small molecule guests: glycine anhydride morphing into glycolide (hence NH → O). I’d like to believe the ligandHybrid step worked as the merged.itp file has: [atoms] ; nr type resnr residue atom cgnr charge mass typeB chargeB massB (…) 3 n 1 UNK N3 3 -0.574900 14.0100 os -0.395900 16.0000 4 n 1 UNK N4 4 -0.574900 14.0100 os -0.395900 16.0000 (…) 9 hn 1 UNK H9 9 0.333500 1.0080 DUM\_hn 0.000000 1.0080 10 hn 1 UNK H10 10 0.333500 1.0080 DUM\_hn 0.000000 1.0080 (…) I then followed the standard md simulation steps, solvated with spc water model, minimised, equilibrated with temp (with single t coupling group) and pressure (Parinello-Rahman, isotropic), and run the production run over 100 ps. I kept LINCS constraints with order 6 (I wasn’t sure of the effect of changing this) and with FEP: ; Free energy control stuff free-energy = yes init-lambda = 0 delta-lambda = 4e-6 sc-function = gapsys sc-alpha = 0.3 sc-sigma = 0.25 sc-power = 1 sc-coul = yes The minimisation was a bit temperamental, requiring a few steep and then stochastic descent steps but the remainder run fine with no excessive variations in temperature or pressure. I run the entire setup in forward and reverse directions: init-lambda = 0/1 ## I was expecting the free energy to not be overly large and yet the pmx analyse produced: Number of forward (0-\>1) trajectories: 1 Number of reverse (1-\>0) trajectories: 1 Temperature : 298.15 K ``` Crooks Gaussian Intersection ``` * * * CGI: Forward Gauss mean = 120.48 kJ/mol std = 0.00 kJ/mol CGI: Reverse Gauss mean = 120.00 kJ/mol std = 0.00 kJ/mol ## Gaussians too close for intersection calculation → Taking difference of mean values CGI: dG = 120.24 kJ/mol CGI: Std Err (bootstrap:parametric) = 0.00 kJ/mol CGI: Std Err (bootstrap) = 0.00 kJ/mol Forward: gaussian quality = nan —\> KS-Test Ok Reverse: gaussian quality = nan —\> KS-Test Ok The remainder of estimators give the same dG value. Firstly, is the proximity of Gaussians a problem? Secondly, what could be the source of such a large value? Great thank you in advance, Katarzyna Zator ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [August 14, 2022, 12:08pm UTC](https://ask.bioexcel.eu/t/pmx-rbfe-gives-oddly-large-deltag/3847/2 "2022-08-14T12:08:15Z")
Several observations: 1. you only ran one transition in the forward and one in the reverse direction. This will not suffice. I recommend to increase the number of transitions to the order of ~100. See chapter 6 in this book chapter for the description of the non-eq protocol: [http://www3.mpibpc.mpg.de/groups/de\_groot/pdf/Gapsys\_MolMod\_2015.pdf](http://www3.mpibpc.mpg.de/groups/de_groot/pdf/Gapsys_MolMod_2015.pdf) 2. so far you have calculated only dG of one leg in the thermodynamic cycle. To get ddG you also need the other leg. Vytas