RMSD Analysis for Protein-ligand-POPC Membrane System

GROMACS version: 2024.2
GROMACS modification: No
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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.


Is there anything that looks wrong?