Usually the initial structure is a good reference..

The initial MD setup structure is good point of reference or the minimized structure could also be used as a reference during RMSD calculations.

Debarati Dasgupta

Thank you, but the result isn't the same if I used the minimized structure compared to the initial structure so which is more accepted as a reference structure?

Hi Sam Mohel I have always mainly used the initial setup.pdb structure for my RMSD reference structure, but even with using the minimized structure it should ideally not deviate too much ( RMSD fluctuations should not > 2Angstroms when using reference structure= minimized pdb file) If your RMSDs are changing too much have a good look at your minimization overlay the structure and inspect what structural changes are causing this? Also try to keep loops out from your RMSD alignment as having too many loops in your protein can cause local RMSD of that region to fluctuate heavily.

Thanks! Post more questions if you have, happy to help!