All Gromacs analysis tools allow you to exclude whatever frames you like with -b and/or -e to set beginning and end times for analysis. This is a simple first step in assessing convergence.

The answer to your question depends on the answers of several more questions:

1. Do you know what RMSF is and understand how it is calculated?

2. What group(s) did you consider for the calculation? Did you carry out the calculation per atom or per residue?

3. Did you eliminate frames from the beginning of the trajectory to adequately account for equilibration? RMSF can be heavily impacted by these early frames if you are not careful.

4. What are you hoping to achieve by measuring RMSF? What is your hypothesis and how does RMSF fit into its evaluation?

Dear Justin Lemkul like your answer and it very helpful

will you please elaborate the third point. and how i will eliminate the starting frames

All Gromacs analysis tools allow you to exclude whatever frames you like with -b and/or -e to set beginning and end times for analysis. This is a simple first step in assessing convergence.

Thank you very much for your helpful answer DR(justin lemkul)

gmx rmsf -f md.xtc -s md.tpr -q protein.pdb -o rmsf.xvg

This command is used for RMSF analysis