# PBC leads to high ligand RMSD after unbinding

**URL:** <https://gromacs.bioexcel.eu/t/pbc-leads-to-high-ligand-rmsd-after-unbinding/10462>\
**Category:** User discussions\
**Tags:** analysis-tools\
**Created:** [October 31, 2024, 6:21pm UTC](https://gromacs.bioexcel.eu/t/pbc-leads-to-high-ligand-rmsd-after-unbinding/10462 "2024-10-31T18:21:44Z")\
**Posts on this page:** 1\
**Page:** 1

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**Author:** ![pchinestrad](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/pchinestrad/32/1465_2.png) [@pchinestrad](https://gromacs.bioexcel.eu/u/pchinestrad)\
**Post date:** [October 31, 2024, 6:21pm UTC](https://gromacs.bioexcel.eu/t/pbc-leads-to-high-ligand-rmsd-after-unbinding/10462/1 "2024-10-31T18:21:44Z")

</div>

GROMACS version: 2024  
GROMACS modification: No

Hi everyone!

I’ve run a 100 ns simulation of a Protein-Ligand complex. I used the Charmm36 forcefield, prepared my protein with CharmmGUI to correctly deal with a Heme that the protein has, and finally prepared my ligand with CgenFF. The simulation ran fine, but I am having issues with the analysis now.

Here are the plots for the interaction energy and the ligand RMSD after fitting the protein backbone.

 ![interaction_energy](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/0/04980d1753f517e864f7bdcb8bc94b9895aa0e56.png)  
 ![rmsd_lig](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/0/003e9c1d6a64367b4a32a9ef29c9855e1bd7e87c.png)

As the plot shows, at first the ligand is bound to the pocket (Purple ligand on the right in the picture below). Then, the RMSD of the ligand spikes to 7 nm at the 60 ns mark, which is the unbinding event, however the 7 nm I believe is a periodic shift, which I was able to confirm by checking the frames in PyMOL (White ligand, left). The ligand then returns and binds near to the initial binding region (Yellow, right), before a second unbinding event ocurrs at around 85 ns. This time however, instead of returning to the pocket, it binds to the other side of the protein (Green, left).

 ![ligand_positions](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/2/2cc3d73cfacf8dd5312f2c7255bc5b25e0f87f91.jpeg)

While the unbinding itself is simply a result, the second binding to the other side of the protein is an artifact, right? Should I do something to avoid this “artifact”? If so, what can I do? I read the forum and found a suggestion to use trjconv with -pbc nojump but given the ligand interacts with the protein in this other region, it is not useful. Maybe make a bigger box? I used a dodecahedron for the box with the distance -d of 1.0.

Any help or tip on how to fix this is greatly appreciated!