# High RMSD value and Fluctuations **URL:** \ **Category:** User discussions\ **Created:** [March 20, 2023, 4:16pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008 "2023-03-20T16:16:34Z")\ **Posts on this page:** 20\ **Page:** 1
**Author:** ![harunalcakan](https://avatars.discourse-cdn.com/v4/letter/h/df705f/32.png) [@harunalcakan](https://gromacs.bioexcel.eu/u/harunalcakan)\ **Post date:** [March 20, 2023, 4:16pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/1 "2023-03-20T16:16:34Z")
GROMACS version: 2019.3 GROMACS modification: No Dear all, I know that there are lots of topics about high RMSD value, sudden jumps or fluctuations. I’ve been scrolling through the topics and trying to apply several trjconv commands into my system but could not even get a smooth RMSD value and I am not sure if my system has crashed. I have carried out protein+ligand MD simulation and I had smooth RMSD values so far except the last system I set up for my simulation. I couldn’t fix my RMSD result even though I have tried several commands that I have listed out of order. > gmx trjconv -f md\_0\_1.xtc -s md\_0\_1.tpr -center -pbc mol > trjconv -s md\_0\_1.tpr -f md\_0\_1.xtc -o md\_0\_1\_fit-rot\_trans.xtc -ur compact -fit rot+trans > gmx trjconv -s prod\_md.tpr -f prod\_md.xtc -o prod\_md\_nopbc\_cluster.xtc -pbc cluster Sadly, these commands did not work, I think I am missing some points since I’m new to GROMACS. Sorry for bringing this topic up again but I got quite confused. Here is the RMSD result of my system: ![Screenshot_3](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/1/1915641d406d7d6829f3bd53231efd097af46a72.png) ---
**Author:** ![harunalcakan](https://avatars.discourse-cdn.com/v4/letter/h/df705f/32.png) [@harunalcakan](https://gromacs.bioexcel.eu/u/harunalcakan)\ **Post date:** [March 22, 2023, 11:37am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/2 "2023-03-22T11:37:18Z")
Would anyone suggest any solution of this problem? Any help would be appreciated. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [March 22, 2023, 1:01pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/3 "2023-03-22T13:01:04Z")
What happens when you watch the trajectory? It looks like there is a legitimate, steady increase but with a few dips. What is happening in the frames with the sudden dips? ---
**Author:** ![harunalcakan](https://avatars.discourse-cdn.com/v4/letter/h/df705f/32.png) [@harunalcakan](https://gromacs.bioexcel.eu/u/harunalcakan)\ **Post date:** [March 23, 2023, 2:39pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/4 "2023-03-23T14:39:46Z")
Hi Mr. Jalemkul First trajectory image belongs to the original .xtc file: ![Screenshot_13](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/7/74216fc9ea91b6ea56b749fbee04c5d387d97564.png) And the second one belongs to the .xtc file that I have modified with above-mentioned commands. ![Screenshot_14](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/5/5d7a7399423e07b5a90082a9096148499e520e9e.png) It seems that my ligand is not within the protein and the commands did not work. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [March 26, 2023, 5:10pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/5 "2023-03-26T17:10:00Z")
Both frames appear to be simple PBC issues. What group are you choosing for the `trjconv` selections related to centering and fitting? You should be choosing the protein, or perhaps just the backbone. If you do, the ligand will wrap to the protein. ---
**Author:** ![Bart](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/bart/32/1389_2.png) [@Bart](https://gromacs.bioexcel.eu/u/Bart)\ **Post date:** [April 6, 2023, 11:08am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/6 "2023-04-06T11:08:42Z")
You can take a look at some code we developed to fix these problems by ensuring assemblies are whole. ([GitHub - BartBruininks/mdvwhole: Density based object completion over PBC.](https://github.com/BartBruininks/mdvwhole)). The selection you would need would be a selection which includes both the protein and the ligand. If you try it and you have any issues feel free to get in contact with me. Cheers, Bart ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 13, 2023, 2:18am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/7 "2023-04-13T02:18:53Z")
Hi, @jalemkul I have same type of issues with my all simulations. Ligand/peptide shows a lot of movement. Rather vibrating at the docked site, it dances outside the protein periphery. Is it normal in MD simulations? or there is something wrong in MDP files? or Ligand didn’t bind strongly/close enough in the docked complex? After > gmx mdrun -deffnm md\_0\_10 I have recentered and performed rotational and translational fitting > gmx trjconv -s md\_0\_10.tpr -f md\_0\_10.xtc -o md\_0\_10\_center.xtc -center -pbc mol -ur compact > gmx trjconv -s md\_0\_10.tpr -f md\_0\_10\_center.xtc -o md\_0\_10\_fit.xtc -fit rot+trans Any suggestions/guidance will be appreciated. Please have a look in the MD movie [https://drive.google.com/file/d/1f75WcRbMkLDoWq9xR7sbQvGsvF1wzHlv/view?usp=share\_link](https://MD_movie) Ligand and the docked sites are in Red and Cyan color respectively. Please find my MDP files attached [production.mdp](https://gromacs.bioexcel.eu/uploads/short-url/lg8HDCh2a9nwgnrxYPy1PRa9nlV.mdp) (2.4 KB) [eq1-nvt.mdp](https://gromacs.bioexcel.eu/uploads/short-url/pGmkFXM7YINYKl4Bf4aGDznhnLV.mdp) (2.8 KB) [eq2-npt.mdp](https://gromacs.bioexcel.eu/uploads/short-url/i36Fm7kAKwAEk9KgaAU1qiyjXSy.mdp) (2.7 KB) [ions.mdp](https://gromacs.bioexcel.eu/uploads/short-url/78mPo86fXmkuXi5kEP08cUmSkO4.mdp) (1.0 KB) [minim.mdp](https://gromacs.bioexcel.eu/uploads/short-url/h8n7HC6rezHTNgYLu1Jp9SoPbhx.mdp) (1.1 KB) ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 13, 2023, 4:53pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/8 "2023-04-13T16:53:33Z")
> [@shivam1](#): > > Is it normal in MD simulations? or there is something wrong in MDP files? or Ligand didn’t bind strongly/close enough in the docked complex? Your ligand clearly dissociates and it isn’t an artifact. What is the ligand and how did you parametrize it? It looks rather small and very flexible, so it will be extremely sensitive to the quality of the topology you generate for it. ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 14, 2023, 12:38am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/9 "2023-04-14T00:38:02Z")
@jalemkul Thanks for kind consideration. Ligand is Gama Amino Butyric Acid ([https://pubchem.ncbi.nlm.nih.gov/compound/119](https://GABA_pubchem)). I used SwissParam Server ([https://www.swissparam.ch/](https://swiss_param)) to generate its PDB from mol2 file and then used `gmx editconf -f LIG.pdb -o LIG.gro` to generate it’s gro file. `out of the scope question:` PDB must be renumbered before MD simulation? Also, chain must not have any missing residues Thanks ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 14, 2023, 2:06am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/10 "2023-04-14T02:06:26Z")
Can you post the topology that it generated? That is a simple molecule so the topology should be straightforward. > [@shivam1](#): > > PDB must be renumbered before MD simulation? No. > [@shivam1](#): > > Also, chain must not have any missing residues Correct. ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 14, 2023, 3:31am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/11 "2023-04-14T03:31:15Z")
Thank you @jalemkul please find the latest Topol file attached. Also find the other LIG associated files in the drive ([https://drive.google.com/file/d/1sfuIDwD\_YpjZsN8QZg2aaS1rkbfsrXZy/view?usp=share\_link](https://LIG.files)). [topol.top](https://gromacs.bioexcel.eu/uploads/short-url/ugJUuzaMpJJITI4c6gEOwb7XUBe.top) (1.7 MB) ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 14, 2023, 12:34pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/12 "2023-04-14T12:34:56Z")
The Google Drive link is inaccessible. The information in topol.top is not terribly useful because what I need is gaba\_lig.itp. ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 14, 2023, 1:31pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/13 "2023-04-14T13:31:35Z")
Please find the link for the gaba\_lig.itp. Can’t upload here as this portal doesn’t allow us to upload certain files. > **[gaba\_lig.itp](https://drive.google.com/file/d/1NyUGg7-ow8ZwzOkN0gbzPWizUR8VqQsx/view?usp=share_link)** > > Google Drive file. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 14, 2023, 2:47pm UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/14 "2023-04-14T14:47:29Z")
That topology makes no sense at all. The charges are totally wrong, with most of the C and H atoms bearing zero charge. The charged amino and carboxy groups are over-polarized with respect to typical CHARMM groups so altogether it probably renders the molecule wildly imbalanced and prone to dissociation. You can generate a topology for this species very easily by analogy from existing CHARMM parameters (by analogy to LYS and GLU, from CHARMM36): ```auto [atoms] ; nr type resnr resid atom cgnr charge mass 1 NH3 1 LIG N 1 -0.3000 14.0067 2 CT2 1 LIG CA 2 0.2100 12.0110 3 CT2 1 LIG CB 3 -0.1800 12.0110 4 CT2 1 LIG CG 4 -0.2800 12.0110 5 CC 1 LIG CD 5 0.6200 12.0110 6 OC 1 LIG OE1 6 -0.7600 15.9994 7 OC 1 LIG OE2 7 -0.7600 15.9994 8 HA2 1 LIG HA2 8 0.0500 1.0079 9 HA2 1 LIG HA3 9 0.0500 1.0079 10 HA2 1 LIG HB2 10 0.0900 1.0079 11 HA2 1 LIG HB3 11 0.0900 1.0079 12 HA2 1 LIG HG2 12 0.0900 1.0079 13 HA2 1 LIG HG3 13 0.0900 1.0079 14 HC 1 LIG H1 14 0.3300 1.0079 15 HC 1 LIG H2 15 0.3300 1.0079 16 HC 1 LIG H3 16 0.3300 1.0079 ``` ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 20, 2023, 1:44am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/15 "2023-04-20T01:44:10Z")
@jalemkul Thanks for your kind response I have generated ligand topology as per your protocol using ‘[https://cgenff.umaryland.edu/userAccount/userLogin.php’](https://cgenff.umaryland.edu/userAccount/userLogin.php%E2%80%99) and cgenff\_charmm2gmx\_py3\_nx2.py script. it still disassociates after some time. Total MD run is 60 ns. Please find the associated files in the drive. [https://drive.google.com/drive/folders/1iqwPVY1OQ\_3Ygfr1p-1K\_enPioIoVPTq?usp=share\_link](https://drive.google.com/drive/folders/1iqwPVY1OQ_3Ygfr1p-1K_enPioIoVPTq?usp=share_link) ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 20, 2023, 2:24am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/16 "2023-04-20T02:24:07Z")
Why are you protonating the carboxylate group? Only under extremely acidic conditions would this be the case. ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 20, 2023, 2:39am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/17 "2023-04-20T02:39:42Z")
Thanks for your kind consideration. I did it as a part of molecular docking protocol where I have to add hydrogen. Not sure though if I can avoid protonation of terminal carboxylate while adding polar hydrogen. Please guide me over it. Thanks ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 20, 2023, 2:50am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/18 "2023-04-20T02:50:55Z")
You need to model real conditions, and GABA is a zwitterion at physiological pH. So you need to protonate it properly, or the model lacks value. ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [April 20, 2023, 2:57am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/19 "2023-04-20T02:57:49Z")
Thank you so much :) Can I use `pbd2pqr` module for the protonation? ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [April 20, 2023, 3:18am UTC](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008/20 "2023-04-20T03:18:11Z")
Sure, there are any number of tools that can accomplish this task. Use whichever one you’re most comfortable with. [Next page](https://gromacs.bioexcel.eu/t/high-rmsd-value-and-fluctuations/6008.md?page=2)