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Root Mean Square Deviation and Fluctuation (RMSD/RMSF)

Introduction

Root-mean-square deviation (RMSD) is a measure of how much a structure differs from a starting or reference structure, usually reported in angstroms (Å) or nanometers (nm). In GROMACS, RMSD is calculated with gmx rms.

Plotting RMSD against time tells us two things:

  1. How much the protein structure has changed since the start of the simulation. Greater RMSD values indicate greater change, and vice versa.
  2. Whether our simulation has achieved convergence, or a state of equilibrium.
Example of RMSD graph with 3 replicates. RMSD is plotted as nanometer (nm) versus time (ns).
RMSD graph from one set of simulations. RMSD is plotted as nanometer (nm) versus time (ns).

Similarly, root-mean-square fluctuation (RMSF) allows us to identify regions that are highly flexible throughout the simulation. RMSF is calculated using gmx rmsf and is plotted as a function of protein residue.

Example of RMSF graph with 3 replicates. RMSF is plotted as nanometer (nm) versus residue number.
RMSF graph from the same simulations. RMSF is plotted as nanometer (nm) versus residue number.

You should have the following files for RMSD/RMSF:

  • Trajectory files (.xtc)
  • Topology file (.tpr)
  • Index files (.ndx) - OPTIONAL

Using gmx rms

To perform RMSD analysis:

gmx rms -f trajectory.xtc -s topology.tpr -o rmsd.xvg
Other options you may find useful
  • -n: index file to be used (see Creating Index Files)
  • -tu: time units (default is ps)
  • -b and -e: the frames to begin and end
  • -skip: number of frames to skip, useful for large simulations

You will be prompted to select two groups, one for least squares fit and one for the RMSD calculations. Select Backbone for both of these.

This will output an XVG file that can be visualized using Grace or Python.

Using gmx rmsf

To perform RMSF analysis:

gmx rmsf -f trajectory.xtc -s topology.tpr -o rmsf.xvg -res
Other options you may find useful
  • -n: index file to be used (see Creating Index Files)
  • -b and -e: the frames to begin and end
  • -skip: number of frames to skip, useful for large simulations

The -res option is required to calculate the average fluctuation for each residue. Select Backbone when prompted.

This will output an XVG file that can be visualized using Grace or Python.

Additional Resources