# Getting very high RMSD value after 75ns

**URL:** <https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138>\
**Category:** User discussions\
**Tags:** mdrun, analysis-tools\
**Created:** [January 24, 2025, 8:08am UTC](https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138 "2025-01-24T08:08:38Z")\
**Posts on this page:** 5\
**Page:** 1

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**Author:** ![nilanjana88](https://avatars.discourse-cdn.com/v4/letter/n/59ef9b/32.png) [@nilanjana88](https://gromacs.bioexcel.eu/u/nilanjana88)\
**Post date:** [January 24, 2025, 8:08am UTC](https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138/1 "2025-01-24T08:08:38Z")

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GROMACS version: 2024.2  
GROMACS modification: No

I am running a protein-ligand md simulation for 100ns. The RMSD plot shows, complex remains stable upto 75ns, but exhibits very high RMSD thereafter. Upon visualizing the trajectory, I observed the ligand is no longer binding to the protein beyond 75ns, and jumping around the simulation box.

I recentered the trajectory using the following command,  
gmx trjconv -s MD.tpr -f MD.xtc -o MD\_center.xtc -center -pbc mol -ur compact  
After recentering, I calculated RMSD, whose plot is attached below.

 ![Screenshot from 2025-01-24 13-31-17](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/3/35c5f594e12c60a0f96df76dcab6e925b6b8559f.png)

I also removed the periodic boundary condition with **pbc -nojump** flag which gave a similar plot.  
I am worried what could’ve gone wrong during the simulation?  
I performed the docking in autodock vina and took the best pose with lowest binding energy, and followed every step in the tutorial [link](http://www.mdtutorials.com/gmx/complex/01_pdb2gmx.html)

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**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\
**Post date:** [January 24, 2025, 8:55am UTC](https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138/2 "2025-01-24T08:55:01Z")

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For how long do you expect the ligand to be bound in a single observation?

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<div class="post-metadata">

**Author:** ![nilanjana88](https://avatars.discourse-cdn.com/v4/letter/n/59ef9b/32.png) [@nilanjana88](https://gromacs.bioexcel.eu/u/nilanjana88)\
**Post date:** [January 24, 2025, 10:09am UTC](https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138/3 "2025-01-24T10:09:49Z")

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Thank you for your response.

I was under the impression that the entire system (protein + ligand) should remain stable throughout the simulation, with an RMSD around 0.3–0.4 (in nm) indicating stability.

Is this kind of behavior typical in protein-ligand simulations? Would it be advisable to rerun the simulation to check if the same pattern repeats?

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<div class="post-metadata">

**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\
**Post date:** [January 24, 2025, 10:20am UTC](https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138/4 "2025-01-24T10:20:06Z")

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Protein-ligand binding is a dynamic process (in almost all cases). You would expect the ligand to leave the binding site (after a while) and rebind again later if you simulate long enough.

The binding kinetics (on/off rates) are different from one protein-ligand complex to another. In some cases it might be difficult to simulate long enough to see the unbinding event. The binding event often takes even longer in a simulation due to the larger volume of exploration.

It is always a good idea to re-run simulations (at least three replicas, but preferably five or more), unless you have a very good reason not to. The observations from one single simulation does not say much about the probabilities or time scales of an event.

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<div class="post-metadata">

**Author:** ![nilanjana88](https://avatars.discourse-cdn.com/v4/letter/n/59ef9b/32.png) [@nilanjana88](https://gromacs.bioexcel.eu/u/nilanjana88)\
**Post date:** [January 24, 2025, 11:39am UTC](https://gromacs.bioexcel.eu/t/getting-very-high-rmsd-value-after-75ns/11138/5 "2025-01-24T11:39:03Z")

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Thank you for your explanations.  
Yes that makes sense. I will consider rerunning the simulation with same force field and parameters to gather more reliable insights.