# Ligand dissociation from the binding pocket and high RMSD (\>8 Å) during a 200 ns MD simulation using

**URL:** <https://gromacs.bioexcel.eu/t/ligand-dissociation-from-the-binding-pocket-and-high-rmsd-8-a-during-a-200-ns-md-simulation-using/13783>\
**Category:** Uncategorized\
**Tags:** analysis-tools\
**Created:** [October 21, 2026, 9:17am UTC](https://gromacs.bioexcel.eu/t/ligand-dissociation-from-the-binding-pocket-and-high-rmsd-8-a-during-a-200-ns-md-simulation-using/13783 "2026-10-21T09:17:13Z")\
**Posts on this page:** 1\
**Page:** 1

<div class="post-metadata">

**Author:** ![R\_Thivarsha](https://avatars.discourse-cdn.com/v4/letter/r/dfb087/32.png) [@R\_Thivarsha](https://gromacs.bioexcel.eu/u/R_Thivarsha)\
**Post date:** [October 21, 2026, 9:17am UTC](https://gromacs.bioexcel.eu/t/ligand-dissociation-from-the-binding-pocket-and-high-rmsd-8-a-during-a-200-ns-md-simulation-using/13783/1 "2026-10-21T09:17:13Z")

</div>

### Background

Hello everyone,

I am performing molecular dynamics (MD) simulations of a protein–ligand complex using GROMACS. I prepared the system using CHARMM-GUI and carried out energy minimization, equilibration, and production MD.

However, during trajectory analysis, I observed that the ligand gradually moves away from the protein’s binding pocket. The RMSD also appears unstable and exceeds 8 Å. I would like to understand the possible reasons for this behavior and determine whether my simulation protocol or analysis procedure needs to be corrected.

### System preparation and simulation protocol

- **System:** Protein–ligand complex

- **System preparation:** CHARMM-GUI

- **MD software:** GROMACS

- **Hardware:** NVIDIA GPU

- **Energy minimization:** Performed using the CHARMM-GUI-generated minimization parameters

- **Equilibration:** Performed using the CHARMM-GUI-generated equilibration parameters

- **Production MD:** 200 ns

- **Force field:** The force field specified in my CHARMM-GUI-generated topology

- **Ligand parameters:** Generated as part of the CHARMM-GUI preparation workflow

I used the following general workflow.

**1. Energy minimization**

```bash
gmx grompp -f step4.0_minimization.mdp \
-c step3_input.gro \
-r step3_input.gro \
-p topol.top \
-o em.tpr

gmx mdrun -v -deffnm em

```

**2. Equilibration**

```bash
gmx grompp -f step4.1_equilibration.mdp \
-c em.gro \
-r step3_input.gro \
-p topol.top \
-o eq1.tpr

gmx mdrun -v -deffnm eq1

```

**3. Production MD**

I modified the number of MD steps in `step5_production.mdp` and generated the production input using:

```bash
gmx grompp -f step5_production.mdp \
-c eq1.gro \
-t eq1.cpt \
-p topol.top \
-o md.tpr

```

I then ran the simulation using:

```bash
gmx mdrun -v -deffnm md \
-nb gpu -pme gpu -pin on -cpt 10

```

The production trajectory was subsequently used for structural analysis.

### Trajectory processing and RMSD analysis

I processed the trajectory to handle periodic boundary conditions and center the system:

```bash
gmx trjconv -s md.tpr \
-f md.xtc \
-o md_noPBC.xtc \
-pbc mol -center

```

I then calculated the protein RMSD using:

```bash
gmx rms -s md.tpr \
-f md_noPBC.xtc \
-n index_new.ndx \
-o rmsd_protein.xvg

```

I also attempted to calculate the ligand RMSD using the ligand and protein groups in the index file.

### Observations

1. The ligand appears to move away from its initial binding pocket during the production simulation.

2. The RMSD profile is not stable and reaches values greater than 8 Å.

3. The ligand does not remain in its initial binding position throughout the simulation.

4. I would like to determine whether the observed behavior reflects genuine ligand dissociation or could be influenced by trajectory processing, RMSD reference selection, or other methodological issues.

### Questions for the community

1. What are the most common causes of ligand dissociation during a protein–ligand MD simulation prepared using CHARMM-GUI?

2. Could inadequate equilibration, incorrect ligand parameters, force-field incompatibility, or issues with the initial binding pose contribute to this behavior?

3. Is an RMSD greater than 8 Å necessarily indicative of an unstable protein–ligand complex, or could it result from the choice of reference structure, atom selection, or trajectory alignment?

4. What is the recommended procedure for calculating protein backbone RMSD and ligand RMSD relative to the initial binding pose?

5. Would it be useful to inspect the equilibration protocol and the ligand topology before attempting another simulation?

I would appreciate suggestions on how to troubleshoot this issue systematically and determine whether the observed ligand dissociation is physically meaningful or related to the simulation setup or analysis procedure.

Thank you for your time and help.