# Oscillational period warning during tripeptide A2I forward simulation **URL:** \ **Category:** pmx\ **Created:** [December 21, 2022, 10:20am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034 "2022-12-21T10:20:41Z")\ **Posts on this page:** 15\ **Page:** 1
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [December 21, 2022, 10:20am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/1 "2022-12-21T10:20:42Z")
Hello, I’m trying to perform forward and reverse simulations for Mutation free energy calculations. I have successfully calculated the total ddG of several tripeptides. They output similar ddG values to pmx webserver. However, there is some tripeptide that cannot be successfully simulated for either forward or reverse simulation. In this tripeptide A2I case, reverse simulation has succeeded, but the forward gave me these two warnings during the NPT equilibration: > WARNING 1 [file posre\_tripep\_A2I.itp, line 10]: > Some parameters for bonded interaction involving perturbed atoms are > specified explicitly in state A, but not B - copying A to B > WARNING 2 [file tripep\_A2I.top, line 17]: > The bond in molecule-type Protein\_chain\_X between atoms 29 DCD1 and 30 > DHD1 has an estimated oscillational period of 8.3e-03 ps, which is less > than 5 times the time step of 2.0e-03 ps. > Maybe you forgot to change the constraints mdp option. I’m using GROMACS v2022 and repeating the forward and reverse simulation 5 times for each protein. But all of the outputs of the forward simulations have the same error. I also get the same error for other several tripeptides. Is this related to the preprocessing or GROMACS version? I took the predetermined itp and topology files for the tripeptide from the pmx webserver. Thank you ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [December 21, 2022, 12:25pm UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/2 "2022-12-21T12:25:38Z")
You seem to be using the older python2 based pmx version. There you will need to constrain all-bonds. If you want to constrain only hydrogen containing bonds, use the python3 based pmx version which you can find in the ‘develop’ branch. This will resolve the oscillation period warning. ---
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [December 26, 2022, 8:07am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/3 "2022-12-26T08:07:23Z")
Hello, thank for your reply. I do use python3 based pmx version from the ‘develop’ branch. I also have tried to reinstall the pmx and did the same protocol but still get the oscillation warning. Is it ok to just ignore the warning? ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [December 27, 2022, 9:49am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/4 "2022-12-27T09:49:52Z")
No, you shouldn’t ignore this warning. The A2I mutation in the ‘amber99sb-star-ildn-mut.ff’ and ‘charmm36m-mut.ff’ forcefields in the ‘develop’ branch doesn’t have DHD1 atom, thus the warning that you see will not be triggered. Could you try ‘develop’ branch with one of these forcefields? ---
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [January 4, 2023, 7:57pm UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/5 "2023-01-04T19:57:00Z")
Thank you for your replies, it helps me a lot. Now I’m performing double system-single box simulation for I2M mutation. The system consisted of the folded\_wt (I2M) and unfolded\_mutant (tripeptide M2I) separated at least 3 nm which solvated and neutralized in a dodecahedron box. I generated the ACE-GLY-MET-GLY-NME capped tripeptide using tleap program and amber99sb-star-ildn force field. I do use the python 3 pmx ‘develop’ branch to mutate both the folded\_wt and unfolded\_mutant pairs. However, I always face these warnings and notes during nvt, npt, and equilibirum simulations: > WARNING 1 [~/dssb\_mdp/eqA/nvt-A.mdp, line 62]: > You are using soft-core interactions while the Van der Waals interactions > are not decoupled (note that the sc-coul option is only active when using > lambda states). Although this will not lead to errors, you will need much > more sampling than without soft-core interactions. Consider using > sc-alpha=0. > > NOTE 1 [file ~/dssb\_mdp/eqA/nvt-A.mdp]: > With PME there is a minor soft core effect present at the cut-off, > proportional to (LJsigma/rcoulomb)^6. This could have a minor effect on > energy conservation, but usually other effects dominate. With a common > sigma value of 0.34 nm the fraction of the particle-particle potential at > the cut-off at lambda=0.5 is around 3.9e-05, while ewald-rtol is 1.0e-05. > > Generating 1-4 interactions: fudge = 0.5 > > WARNING 2 [file posre\_Protein\_chain\_A.itp, line 233]: > Some parameters for bonded interaction involving perturbed atoms are > specified explicitly in state A, but not B - copying A to B > > NOTE 2 [file newtop.top, line 25]: > The bond in molecule-type Protein\_chain\_X between atoms 18 CB and 31 DCG2 > has an estimated oscillational period of 1.9e-02 ps, which is less than > 10 times the time step of 2.0e-03 ps. > Maybe you forgot to change the constraints mdp option. > > Number of degrees of freedom in T-Coupling group Protein is 6647.00 > Number of degrees of freedom in T-Coupling group non-Protein is 126471.00 > > NOTE 3 [file ~/dssb\_mdp/eqA/nvt-A.mdp]: > Removing center of mass motion in the presence of position restraints > might cause artifacts. When you are using position restraints to > equilibrate a macro-molecule, the artifacts are usually negligible. > > There were 3 notes > > There were 2 warnings I think Warning\_1 and Note\_1 can be ignored as described by the book. But how about the Warning\_2 and (especially) Note\_2? - Warning\_2, posre\_Protein\_chain\_A.itp, line 233: > 453 1 1000 1000 1000 - Note 2: I think DCG2 atom for M2I exists in ‘develop’ branch and not in ‘master’ but why I still get oscillational period messages? And also I have a question about the soft-core function. Most of the existing examples are using the sc parameters for sc-function=beutler. As now the GROMACS 2022 version supports sc-function=gapsys. Is it better to use this function instead? ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [January 5, 2023, 10:09am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/6 "2023-01-05T10:09:25Z")
Yes, I see now what is causing it. The default value for “–scale\_mass” flag is set too low (currently it is 0.25). This flag tells to scale down dummy atom masses by multiplying the mass of the real atom in another state by the scaling factor. Such scaling helps with simulation stability for the perturbations where many dummies need to be introduced. However, scaling also cannot be too low, because it will trigger the Notes about too frequent vibration period that you see now. Ideally, I would need to check for the oscillation period within pmx, but as a quick fix for now I increased this scaling factor to 0.33. I pushed this change to the ‘develop’ branch. Regarding sc-function, in many cases both functions will yield identical free energy estimates, so for now you could as well continue with the standard beutler variant. However, if you notice some erratic behavior of the calculated free energies (due to large jumps in the dhdl curves), I would recommend trying out another function with the default parameters. Such problematic cases usually occur for more complex perturbations, e.g. mutations involving charge change. ---
**Author:** ![s.pallav](https://avatars.discourse-cdn.com/v4/letter/s/5f8ce5/32.png) [@s.pallav](https://ask.bioexcel.eu/u/s.pallav)\ **Post date:** [January 6, 2023, 4:34am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/7 "2023-01-06T04:34:24Z")
Hi @vgapsys, Should it be the same case of ligand hybrids where I let the tool to do the thing(default parameters) and do not explicitly provide a number to scale dummy masses or angle parameters or dihedral parameters. Regards, Pallav. ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [January 6, 2023, 9:24am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/8 "2023-01-06T09:24:13Z")
If you observe instabilities, e.g. simulations show LINCS warnings and crash, scaling dummy masses might help. Otherwise, you can continue with the default settings of non-scaled masses. ---
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [January 9, 2023, 8:09pm UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/9 "2023-01-09T20:09:33Z")
Thanks a lot! now it worked smoothly without the oscillational period warning. The soft-core warning has gone because I changed the sc-function to ‘gapsys’. However, I still got the: > WARNING 1 [file posre\_Protein\_chain\_A.itp, line 233]: > Some parameters for bonded interaction involving perturbed atoms are > specified explicitly in state A, but not B - copying A to B Is it totally fine to ignore it? ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [January 10, 2023, 9:32am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/10 "2023-01-10T09:32:45Z")
What interaction is defined on line 233? ---
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [January 10, 2023, 6:53pm UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/11 "2023-01-10T18:53:19Z")
file posre\_Protein\_chain\_A.itp, line 233: > 453 1 1000 1000 1000 atom #453 in pmx\_topol\_Protein\_chain\_A.itp: > 450 O 26 THR O 450 -0.567900 16.0000 > 451 N 27 I2M N 451 -0.415700 14.0100 > 452 H 27 I2M H 452 0.271900 1.0080 > 453 CT 27 I2M CA 453 -0.059700 12.0100 CT -0.023700 12.0100 > 454 H1 27 I2M HA 454 0.086900 1.0080 H1 0.088000 1.0080 > 455 CT 27 I2M CB 455 0.130300 12.0100 CT 0.034200 12.0100 > 456 HC 27 I2M HB 456 0.018700 1.0080 HC 0.024100 1.0080 > 457 CT 27 I2M CG1 457 -0.043000 12.0100 CT 0.001800 12.0100 > 458 HC 27 I2M HG11 458 0.023600 1.0080 H1 0.044000 1.0080 > 459 HC 27 I2M HG12 459 0.023600 1.0080 H1 0.044000 1.0080 > 460 CT 27 I2M CG2 460 -0.320400 12.0100 DUM\_CT 0.000000 3.9633 > 461 HC 27 I2M HG21 461 0.088200 1.0080 DUM\_HC 0.000000 1.0000 > 462 HC 27 I2M HG22 462 0.088200 1.0080 DUM\_HC 0.000000 1.0000 > 463 HC 27 I2M HG23 463 0.088200 1.0080 DUM\_HC 0.000000 1.0000 > 464 CT 27 I2M CD1 464 -0.066000 12.0100 S -0.273700 32.0600 > 465 HC 27 I2M HD11 465 0.018600 1.0080 DUM\_HC 0.000000 1.0000 > 466 HC 27 I2M HD12 466 0.018600 1.0080 DUM\_HC 0.000000 1.0000 > 467 HC 27 I2M HD13 467 0.018600 1.0080 DUM\_HC 0.000000 1.0000 > 468 C 27 I2M C 468 0.597300 12.0100 > 469 O 27 I2M O 469 -0.567900 16.0000 > 470 DUM\_HC 27 I2M HV1 470 0.000000 1.0000 HC 0.024100 1.0080 > 471 DUM\_CT 27 I2M DCE 471 0.000000 3.9633 CT -0.053600 12.0100 > 472 DUM\_H1 27 I2M HV2 472 0.000000 1.0000 H1 0.068400 1.0080 > 473 DUM\_H1 27 I2M HV3 473 0.000000 1.0000 H1 0.068400 1.0080 > 474 DUM\_H1 27 I2M HV4 474 0.000000 1.0000 H1 0.068400 1.0080 ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [January 11, 2023, 8:13am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/12 "2023-01-11T08:13:55Z")
Right, so the perturbed Calpha atom has a position restraint. The posre is defined for stateA only, but since the atom is perturbed it is not clear what position restraint should be in the stateB. In this case Gromacs warns that it will simply use the same force constants for posre in both states. I think this is what you would like to have, so it is safe to ignore this warning. ---
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [January 24, 2023, 2:53pm UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/13 "2023-01-24T14:53:51Z")
I noticed that some of the mutation types need larger values for the --scale\_mass (i.e \> 0.6) to avoid the oscillation warning. However, it seems that larger mass-scale values often cause energy minimization failure. On the other hand, when low/default value (0.33) is outputting an oscillation warning, the MD steps went smoothly through the production step. Then, when I use the default scale and ignore the oscillation warning, the ddG results from double-system single-box simulation on 5 different mutation cases (3 simulations each → 15 simulations in total) show a relatively accurate to experimental value with around \<= 1 kcal/mol difference. Is there any way to check the optimal scale value for each mutation type? In my case, the results show somehow large scale value causes the EM step to fail and the oscillation warning from too low scale-mass value does not really affect the calculated ddG performance. ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [January 30, 2023, 9:13am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/14 "2023-01-30T09:13:05Z")
Masses should have no influence on energy minimization. Are you sure there is nothing else at play there? Maybe it could be related to some energy minimization bug, e.g. [Energy minimisation won't go to the minimum point when GPU is used. (#4533) · Issues · GROMACS / GROMACS · GitLab](https://gitlab.com/gromacs/gromacs/-/issues/4533) You are mentioning a warning about oscillation period with the scaling of 0.33: are you sure this is a warning and not a note? If it is a note, it is safe to ignore it ---
**Author:** ![jasoank](https://avatars.discourse-cdn.com/v4/letter/j/ea666f/32.png) [@jasoank](https://ask.bioexcel.eu/u/jasoank)\ **Post date:** [January 31, 2023, 11:49am UTC](https://ask.bioexcel.eu/t/oscillational-period-warning-during-tripeptide-a2i-forward-simulation/4034/15 "2023-01-31T11:49:15Z")
Yes, you are correct. Sorry it was my misunderstanding. Thanks a lot! The link you gave solved my EM problem. I tried to find solutions for the energy minimization failure problems previously, but only found generic solutions. Since it was a Warning previous GROMACS version (before 2022), I thought it was harmful even if it became a note instead the latest version. I see, so it safe to ignore it. Many many thanks for your kind replies and solutions!