# Strange PMF profile after umbrella sampling of the peptide pulling **URL:** \ **Category:** User discussions\ **Tags:** umbrella-sampling\ **Created:** [January 6, 2024, 5:17am UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984 "2024-01-06T05:17:51Z")\ **Posts on this page:** 8\ **Page:** 1
**Author:** ![Solly](https://avatars.discourse-cdn.com/v4/letter/s/43a26b/32.png) [@Solly](https://gromacs.bioexcel.eu/u/Solly)\ **Post date:** [January 6, 2024, 5:17am UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/1 "2024-01-06T05:17:51Z")
GROMACS version: 2022.3 GROMACS modification: No Dear GROMACS users! I’ve gor a problem with calculating my PMF profile after umbrella sampling of protein-peptide pulling process. I’ve made an umbrella simulation for the complex of the beta-amyloid peptide with a receptor protein and calculated the binding free energy from PMF profile. It was very close to experimental values. Then i’ve thied to do umbrella sampling with the same receptor and modified peptide. I’ve made two isoforms which were shorter than initial. The pulling was ok and the umbrella sampling too as I can see looking at the trajectories. The distribution histograms were overlaping pretty good with no free spacing between them except a couple of small intervals that cannot reflect dramatically the whole profile and can be easily removed with additional md in new windows. But when I’ve tried to build the pmf profile for each of these new systems, I’ve got something weird in both systems. Here i’ve attached the data for one of the peptides. For the second the situation is very similar. I’ve attached the figure of the PMF profile and histo.xvg. Does anybody know how can such strange profile emerge? ![profile](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/e/e11600764675f708c6f72a6d66949f884552884c.jpeg) [histo.xvg](https://gromacs.bioexcel.eu/uploads/short-url/yBiDJKXhLPlrs2yEnmp3oCAsy0Z.xvg) (84.6 KB) My mdp file was : title = Umbrella pulling simulation define = -DPOSRES\_A ; Run parameters integrator = md dt = 0.002 tinit = 0 nsteps = 5000000 ; 10 ns nstcomm = 10 ; Output parameters nstxout-compressed = 5000 ; every 10 ps nstenergy = 5000 ; Bond parameters constraint\_algorithm = lincs constraints = h-bonds continuation = yes ; Single-range cutoff scheme cutoff-scheme = Verlet nstlist = 20 ns\_type = grid rlist = 1.4 rcoulomb = 1.4 rvdw = 1.4 ; PME electrostatics parameters coulombtype = PME fourierspacing = 0.12 fourier\_nx = 0 fourier\_ny = 0 fourier\_nz = 0 pme\_order = 4 ewald\_rtol = 1e-5 optimize\_fft = yes ; Berendsen temperature coupling is on in two groups Tcoupl = V-rescale tc\_grps = Protein Non-Protein tau\_t = 1.0 1.0 ref\_t = 310 310 ; Pressure coupling is on Pcoupl = Berendsen pcoupltype = isotropic tau\_p = 2.0 compressibility = 4.5e-5 ref\_p = 1.0 refcoord\_scaling = com ; Generate velocities is off gen\_vel = no ; Periodic boundary conditions are on in all directions pbc = xyz ; Long-range dispersion correction DispCorr = EnerPres ; Pull code pull = yes pull\_ncoords = 1 ; only one reaction coordinate pull\_ngroups = 2 ; two groups defining one reaction coordinate pull\_group1\_name = abeta pull\_group2\_name = rage pull\_coord1\_type = umbrella ; harmonic potential pull\_coord1\_geometry = distance ; simple distance increase pull\_coord1\_dim = N N Y pull\_coord1\_groups = 1 2 pull\_coord1\_start = yes ; define initial COM distance \> 0 pull\_coord1\_rate = 0.0 ; restrain in place pull\_coord1\_k = 1000 ; kJ mol^-1 nm^-2 pull-group1-pbcatom = 4801 pull-pbc-ref-prev-step-com = yes Thank you in advance for the answers! Best regards, Anna ---
**Author:** ![scinikhil](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/scinikhil/32/3483_2.png) [@scinikhil](https://gromacs.bioexcel.eu/u/scinikhil)\ **Post date:** [January 7, 2024, 1:50am UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/2 "2024-01-07T01:50:25Z")
This is probably poor sampling, you dont have overlaping smooth bell-shaped curve. ![image](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/d/d459a61a741ec56d2d4b0b9c6966adfc69311a56.png) ---
**Author:** ![Solly](https://avatars.discourse-cdn.com/v4/letter/s/43a26b/32.png) [@Solly](https://gromacs.bioexcel.eu/u/Solly)\ **Post date:** [January 19, 2024, 8:37pm UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/3 "2024-01-19T20:37:21Z")
Hello again everybody! I did an additional sampling and now my histogramm looks that way : [histo.xvg](https://gromacs.bioexcel.eu/uploads/short-url/6QhNrHYw6vhfagUYWNoVQ4kFUfg.xvg) (181.1 KB) But again my profile is bad : ![profile](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/9/9e95bc77c99c681cf1efca36b4cc1337ad4fc6e9.jpeg) So I suppose the problem is not in the sampling ---
**Author:** ![scinikhil](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/scinikhil/32/3483_2.png) [@scinikhil](https://gromacs.bioexcel.eu/u/scinikhil)\ **Post date:** [January 19, 2024, 9:21pm UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/4 "2024-01-19T21:21:14Z")
What you are trying to do? what you are pulling? ---
**Author:** ![Solly](https://avatars.discourse-cdn.com/v4/letter/s/43a26b/32.png) [@Solly](https://gromacs.bioexcel.eu/u/Solly)\ **Post date:** [January 20, 2024, 6:15pm UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/5 "2024-01-20T18:15:22Z")
I am attempting to calculate the binding coefficient for the RAGE protein with small peptides. I have successfully achieved this with the beta-amyloid peptide, and the resulting coefficient was close to the experimental value ([IJMS | Free Full-Text | Docking and Molecular Dynamics-Based Identification of Interaction between Various Beta-Amyloid Isoforms and RAGE Receptor](https://doi.org/10.3390/ijms231911816) - my work, including all data on previous umbrella sampling, can be found here). Now, I need to perform umbrella sampling for shorter peptides, specifically halves of the full-length peptide. I am following the same procedure as in the previous successful case. I begin by taking the complex of RAGE and a peptide, namely Aβ17-42, then perform a pulling simulation to extract initial conformations for windows from this trajectory. I then perform umbrella MD for 10 ns, which was sufficient in the previous case. I have checked five random trajectories and they appear fine, with no artifacts. The distributions of the center of mass (COM) are good, as evidenced by my previous message. However, the resulting potential of mean force (PMF) is quite strange, especially considering that it was good for the whole peptide and the same receptor. Thank you for your help, Anna ---
**Author:** ![Solly](https://avatars.discourse-cdn.com/v4/letter/s/43a26b/32.png) [@Solly](https://gromacs.bioexcel.eu/u/Solly)\ **Post date:** [February 20, 2024, 7:18pm UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/6 "2024-02-20T19:18:38Z")
Hello Gromacs users! I still have problems with my PMF graph. I’ve rerun all the md from the very start. I’ve removed pull-group1-pbcatom value from my mdp file. [md\_umbrella.mdp|attachment] (upload://hXnORMuVSpDmQQAJ5DJ12JVGl3P.mdp) (1.9 KB) ![profile](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/1/1cf52f28ccc3d4f4abfa93c17be3df3c7a34630b.jpeg) The plots look better, but the energy range is still to low. Could anybody provide any guidance on how to solve the problem? Best regards, Anna ---
**Author:** ![oSalad](https://avatars.discourse-cdn.com/v4/letter/o/898d66/32.png) [@oSalad](https://gromacs.bioexcel.eu/u/oSalad)\ **Post date:** [February 20, 2024, 11:14pm UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/7 "2024-02-20T23:14:33Z")
What is the experimental dG? You may have a very weak binder. ---
**Author:** ![Solly](https://avatars.discourse-cdn.com/v4/letter/s/43a26b/32.png) [@Solly](https://gromacs.bioexcel.eu/u/Solly)\ **Post date:** [March 5, 2024, 4:08am UTC](https://gromacs.bioexcel.eu/t/strange-pmf-profile-after-umbrella-sampling-of-the-peptide-pulling/7984/8 "2024-03-05T04:08:47Z")
The dissociation constant (Kd) of Aβ42 with the soluble extracellular part of RAGE (sRAGE), determined using microscale thermophoresis (MST), was 1.0 ± 0.2 µM. That corresponds to — 8,171 kcal/mol. The dissociation constant (Kd) of Aβ17-42 was almost the same, while Kd of Aβ1-16 was too small to measure. So I’ve got half the dG value as in the experiment.