MD Analysis

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During the calculation of rmsd or other parameters. I used following command gmx rms -s em.tpr -f md_0_10_center.xtc -n index.ndx -tu ns -o rmsd_jz4.xvg. I have confusion on .tpr file. Is .tpr file should be from the energy minimization step (i.e. em.tpr) or should it be from MD stage (i.e. md.tpr)?

It depends on what you want. The em.tpr file contains the starting structure, prior to any influence of the force field (presumably an experimentally determined structure). If you want RMSD relative to this structure, supply it as the reference. The md.tpr file contains the equilibrated structure, which has been energy-minimized and equilibrated after some period of dynamics. If this RMSD value is useful to you, use that as the reference.

Thank You Sir