# RMSD Analysis for Protein-ligand-POPC Membrane System

**URL:** <https://gromacs.bioexcel.eu/t/rmsd-analysis-for-protein-ligand-popc-membrane-system/11518>\
**Category:** User discussions\
**Tags:** analysis-tools\
**Created:** [February 11, 2025, 12:01pm UTC](https://gromacs.bioexcel.eu/t/rmsd-analysis-for-protein-ligand-popc-membrane-system/11518 "2025-02-11T12:01:11Z")\
**Posts on this page:** 1\
**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:** [February 11, 2025, 12:01pm UTC](https://gromacs.bioexcel.eu/t/rmsd-analysis-for-protein-ligand-popc-membrane-system/11518/1 "2025-02-11T12:01:11Z")

</div>

GROMACS version: 2024.2  
GROMACS modification: No  
Here post your question

Hi,  
I am running MD Simulation for a protein-ligand-POPC membrane system. I have prepared the system using CHARMM GUI, and performed post-MD analysis like RMSD, RMSF to assess equilibration.

I would like to get feedback on whether the steps I’ve followed are correct in order.

The steps I followed:  
For PBC correction,

1. " -pbc whole " to make the molecule whole
2. " -pbc nojump " with the first frame as reference, to prevent jump accross periodic boundaries
3. " -center -ur compact" to center the proteion, selecting “protein” for centering and “system” as output.  
Alternatively,  
Step-3 have been modified to include " -pbc mol -center yes “, or " - fit rot+trans”

But the RMSD plot for protein backbone shows it’s not very stable. It even reaches 1nm for several timepoints, and then decreases to 0.65 nm.

The protein has two subunits, a 7-TM bundle, and a separate intracellular helix which is inactive and not connected to the 7TM part. Upon visualizing the trajectory in VMD, it seems that two subunits are moving apart from each other.  
I tried to cluster two subunits using " -pbc cluster", but got an error.

Below is the RMSD plot I got.

 ![Screenshot from 2025-02-11 17-30-05](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/e/e297b9eba0bc706377261f662f00a9ce3ec9564a.png)  
Is there anything that looks wrong?