How to interpret MD simulation results

What RMSD, RMSF, radius of gyration, SASA, hydrogen bonds, PCA and MM/PBSA tell you, and how to avoid common misreadings.

The main MD analysis metrics

Item	What it is	How to read it
RMSD	Overall deviation from the starting structure over time.	A plateau means the system has equilibrated and is stable.
RMSF	Per-residue flexibility.	Loops are naturally flexible. Check binding-site residues.
Radius of gyration (Rg)	Compactness of the protein.	A stable Rg suggests no unfolding.
SASA	Solvent-accessible surface area.	Large shifts can mean burial or exposure changes.
Hydrogen bonds	Number and persistence of ligand–protein hydrogen bonds.	Persistent bonds support a stable binding mode.
PCA and FEL	Dominant collective motions and the energy landscape.	Shows the conformational states sampled.
MM/PBSA	End-state binding free-energy estimate.	Use for relative ranking of similar ligands.

A sensible reading order

Check the run itself first: temperature, pressure, density and energy should be stable.
Look at protein RMSD, then ligand RMSD relative to the binding site, to see whether the pose is retained.
Use RMSF to find flexible regions and compare binding-site residues.
Confirm compactness with Rg and surface changes with SASA.
Count persistent hydrogen bonds and key contacts across the trajectory.
Use PCA, free-energy landscapes and clustering to understand conformational states.
Estimate binding energy with MM/PBSA on a stable part of the trajectory.

Common mistakes

Drawing conclusions from one short run without checking convergence.
Calculating RMSD before fixing periodic boundary conditions and fitting.
Treating MM/PBSA numbers as exact experimental affinities.
Ignoring ligand drift because the protein RMSD looks stable.

Before this step

If you have not built the simulation yet, read the GROMACS workflow guide.

Frequently asked questions

What is a good RMSD value in MD?

There is no single threshold. For a stable folded protein, backbone RMSD usually plateaus, often within about 1 to 3 Å. The key signal is convergence rather than drift.

What does a high RMSF mean?

It marks flexible regions such as loops and termini. Lower RMSF in the binding site on ligand binding can indicate stabilisation.

What does radius of gyration show?

Compactness. A stable value suggests the protein stays folded, while a steady increase can indicate unfolding or opening.

Is MM/PBSA the same as experimental affinity?

No. It is an approximate estimate best used to rank related compounds.

Build topologies, solvate and neutralise, minimise, equilibrate in NVT then NPT, run production, then correct periodic boundaries before analysis.

Free-energy guideMM/PBSA Binding Free Energy Explained for Beginners

MM/PBSA estimates binding energy from MD snapshots. It ranks similar ligands reasonably well but is not an exact measure of affinity.

Related service

ServiceMD trajectory analysis

Analysis of your existing molecular dynamics trajectories: RMSD, RMSF, radius of gyration, SASA, hydrogen bonds, contacts, PCA, clustering and free-energy landscapes.

Written by Sohel Akhtar Ansari, ReactorMD founder (ORCID). Computational predictions are not experimental confirmation.