GROMACS

What is GROMACS?

GROMACS (GROningen MAchine for Chemical Simulations) is one of the most widely used molecular dynamics engines in computational biology. Originally developed at the University of Groningen in 1991, it has grown into a community-maintained open-source project known for exceptional performance on both CPUs and GPUs.

Molecular dynamics (MD) simulates how atoms move over time by integrating Newton's equations of motion. For proteins, this reveals conformational flexibility, loop motions, domain rearrangements, and stability under different conditions. GROMACS handles the full pipeline: system setup, solvation, energy minimization, equilibration, production dynamics, and trajectory analysis.

How does GROMACS work?

A GROMACS simulation progresses through several stages, each building on the previous one.

Energy minimization

The input structure often contains steric clashes or suboptimal bond geometries from crystallography or homology modeling. Steepest-descent minimization relaxes these high-energy contacts before dynamics begins. The algorithm iteratively adjusts atomic positions to reduce the total potential energy until convergence or a step limit is reached.

NVT equilibration

With clashes resolved, the system is heated to the target temperature. During NVT (constant Number, Volume, Temperature) equilibration, position restraints hold the protein backbone in place while the solvent and ions settle around it. The V-rescale thermostat couples the system to a heat bath, generating a canonical ensemble with correct kinetic energy fluctuations.

NPT equilibration

Once the temperature is stable, the system transitions to NPT (constant Number, Pressure, Temperature) equilibration. A C-rescale barostat adjusts the simulation box volume while sampling the target pressure ensemble. Position restraints remain active during this phase to prevent premature structural drift while the box dimensions adjust.

Production MD

Restraints are released and the system evolves freely. The Parrinello-Rahman barostat is used for production sampling. Coordinates are saved at regular intervals to form the trajectory.

Trajectory analysis

After production, GROMACS analysis commands compute backbone RMSD and RMSF, radius of gyration, secondary structure assignment, solvent-accessible surface area, hydrogen bonds, Ramachandran angles, and an averaged inter-residue distance map. Native outputs are returned alongside display-ready CSV tables.

How to use GROMACS online

Upload a protein structure or fetch an RCSB PDB entry, then choose the simulation duration, force field, and solvent settings. ProteinIQ prepares and equilibrates the system before running GROMACS molecular dynamics online. Results include a final structure, trajectory, selected analyses, and the simulation files needed to inspect the setup.

The Starting point setting also accepts an existing native TPR or a ZIP of native coordinate, topology and MDP files. These modes run GROMACS 2026.0 with the submitted system and parameters, including parameterized ligands, nucleic acids and custom residues. Protein preparation settings and protein-specific analysis selections apply to the protein structure mode.

Runs have a 100-credit minimum. The exact quote is calculated before submission using the selected duration and compute backend. Native inputs use a conservative system-size estimate for automatic routing and quoting; choose CPU or GPU explicitly when appropriate. Credits are metered at the existing CPU or GPU rate while execution runs.

Input

Input	Description
Protein Structure	PDB file upload, mmCIF file, or RCSB PDB ID (e.g., 1UBQ). Maximum 50 MB.
Native GROMACS run input (.tpr)	Complete native run input, maximum 50 MB. Used in Run a native prepared system mode.
Checkpoint files	Optional captured input directory for TPR mode. Preserve relative paths; checkpoint continuation requires md.cpt. Maximum 50 MB compressed and expanded.
Native coordinates, topology and MDP files (.zip)	Complete captured input directory for native file preparation, including every custom force-field and topology include. Maximum 50 MB compressed and expanded.

The structure should contain standard amino acid residues. mmCIF uploads are converted to PDB coordinates before pdb2gmx because GROMACS topology generation does not read mmCIF directly. By default, HETATM records are rejected instead of silently removed, since ligands, cofactors, crystallographic waters, ions, and modified residues change the simulated system. Pre-process structures with PDBFixer if needed, or explicitly choose protein-only HETATM stripping in Advanced settings.

These protein checks apply only to Prepare a protein structure. Native modes retain the complete submitted system. A TPR is passed directly to mdrun; a native ZIP is preprocessed by grompp using the selected files without modifying their contents or injecting protein presets. Custom parameters must already be valid for GROMACS. Missing parameters, unsupported native features and native warnings are reported by GROMACS.

Native file settings

Parameter	Type	Default	Description
MDP file	string	run.mdp	Relative path inside the ZIP passed to grompp -f.
Coordinate file	string	input.gro	Relative path passed to grompp -c.
Topology file	string	topol.top	Relative path passed to grompp -p. Include files retain their original directory layout.
Position-restraint reference file (optional)	string	optional	Relative path passed to grompp -r when supplied.
Index file (optional)	string	optional	Relative path passed to grompp -n when supplied.
Resume checkpoint	boolean	false	Requires md.cpt in the ZIP. Continuation writes a new simulation part with -noappend; a fresh run does not resume a bundled checkpoint.

For native dynamics, Simulation Duration sets the admitted duration bound and quote; it never rewrites the MDP or TPR. The native step count and timestep must specify 1 ns or more and fit the selected bound, up to 200 ns. Native minimization retains the 500 to 5000 step limit. Minimization and normal-mode calculations have no reported physical duration. Temperature, pressure, timestep, constraints and output frequency come from the native inputs. Protein preparation controls are hidden for these modes. Allowed grompp warnings remains an explicit setting with a default of 0 for native preprocessing.

Simulation settings

Setting	Description
Simulation Duration	Production MD length in nanoseconds (1-200 ns, default 10). Longer runs capture slower motions but cost more.
Force Field	Parameterization for interatomic interactions. See force field comparison below. Default: AMBER99SB-ILDN.
Water Model	Explicit solvent model. Available choices are validated against the selected force field. Default: TIP3P.

Environment settings

Setting	Description
Temperature	Simulation temperature (200-400 K, default 300 K). Physiological temperature is 310 K.
Pressure	Target pressure (0.5-2.0 bar, default 1.0 bar).
Ionic Strength	NaCl concentration in molar (0-0.5 M, default 0.15 M). Ions neutralize the system charge plus add excess salt.

Advanced settings

Setting	Description
Timestep	Integration step size. 2 fs is standard with LINCS hydrogen-bond constraints. 4 fs enables GROMACS hydrogen mass repartitioning and requires bond constraints.
Minimization steps	Steepest-descent steps before dynamics (500-5000, default 1000).
Equilibration	Combined NVT + NPT equilibration time (0.1-2.0 ns, default 0.5 ns). Split equally between the two phases.
Bond constraints	H-bonds only (default), All bonds, or None. The 4 fs timestep requires H-bond or all-bond constraints.
Save interval	Trajectory frame spacing. 10 ps (detailed), 50 ps (standard), or 100 ps (compact).
Remove water from output	Strip solvent from the output trajectory to reduce file size. Enabled by default.
Analysis level	Full returns all native analysis files and display CSVs, Standard returns core energy/RMSD/RMSF/Rg/DSSP outputs faster, and None skips post-run analysis while still returning raw GROMACS artifacts.
Compute backend	Auto routes larger or longer jobs to GPU when useful, CPU forces CPU execution, and GPU requests the pinned CUDA runtime on an A10G. GPU failures are reported rather than retried on billed GPU hardware with CPU execution.
HETATM handling	Reject HETATM records by default, or explicitly strip them for protein-only simulations. Rejected or stripped records are included in the artifacts ZIP.
Regenerate hydrogens	Off by default, preserving pdb2gmx behavior. Enable it to ignore input hydrogens and regenerate them from the selected force field.
Chain separation	Passed to pdb2gmx -chainsep for multi-chain structures.
Merge chains	Passed to pdb2gmx -merge; useful only when chains should be one chemical molecule.
Box type	Passed to editconf -bt; default is rhombic dodecahedron.
Box padding	Passed to editconf -d; larger padding increases solvent count and runtime.
Positive ion / Negative ion	Passed to genion -pname and -nname.
Allowed grompp warnings	Passed to grompp -maxwarn only when set above zero. Default is 0, matching GROMACS safer behavior.
Energy terms	Controls which gmx energy terms are extracted to CSV. Raw md.edr is always downloadable.

Output

The simulation produces:

• Final structure: PDB file of the last frame, centered and processed
• Trajectory: XTC file containing all saved frames (protein-only if water removal is enabled)
• Analysis CSVs: Structural and energy metrics computed across the trajectory (see interpreting results below)
• Raw artifacts ZIP: All generated GROMACS topology and include files, MDP, TPR, log, checkpoint, trajectory, energy, analysis, and setup files for reproducibility

Native modes return the complete final system as PDB, unmodified final GRO coordinates, the captured or compiled TPR, and any native XTC/TRR trajectory. The archive retains the original uploaded ZIP, custom includes, original MDP, native energy/checkpoint/log files and full command diagnostics. native-input.mdp is a diagnostic dump of the TPR, not a replacement for the original MDP. Protein-only trimming and protein-specific analysis tables are not applied to native systems. A runtime-limited run retains its available native artifacts without labeling a partial system as a completed final structure.

Force fields

Each force field represents a different parameterization philosophy. The choice affects secondary structure propensities, sidechain dynamics, and solvation behavior.

Force field	Type	Strengths
AMBER99SB-ILDN	All-atom	Well-validated for protein folding and dynamics. Improved sidechain torsions for Ile, Leu, Asp, Asn. Default choice for most applications.
CHARMM27	All-atom	Reliable for proteins and nucleic acids. Tends to slightly favor helical conformations.
GROMOS96 54a7	United-atom	Non-polar hydrogens are implicit, reducing atom count and speeding up simulations. Parameterized against thermodynamic data (heats of vaporization, solvation free energies).
OPLS-AA/L	All-atom	Optimized for liquid-state properties and small-molecule interactions. Useful when solvent thermodynamics matter.

For general protein dynamics, AMBER99SB-ILDN is the most common choice in recent literature. GROMOS96 54a7 offers faster simulations at the cost of losing explicit non-polar hydrogen detail.

Water models

Protein preparation uses explicit solvation. The default is a dodecahedral box with 1.0 nm padding around the solute; Advanced settings also provide cubic and octahedral boxes and configurable padding. Native modes retain the solvent and box supplied in their inputs.

Model	Sites	Notes
TIP3P	3	Standard choice, compatible with many force fields. Diffusion coefficient is higher than experiment.
SPC/E	3	Better self-diffusion and dielectric constant than TIP3P. Includes a polarization correction.
SPC	3	Simpler parameterization. Fastest of the three, but less accurate for dynamical properties.
TIP4P / TIP4P-Ew	4	Available for force fields that support four-site water models.

ProteinIQ prevents unsupported force-field/water combinations. GROMOS96 54a7 is limited to SPC and SPC/E; CHARMM27 does not offer TIP4P-Ew in this runtime. Water model choice can influence conformational sampling and solvation dynamics.

Interpreting results

RMSD

Root mean square deviation measures the displacement of selected atoms after fitting to a reference structure. The backbone RMSD trace is reported in nanometers: 0.2 nm equals 2 Å. Its interpretation depends on the reference, fitting group, protein size, and flexible regions, so there is no universal RMSD cutoff for a successful simulation.

A plateau shows that deviation from the chosen reference has leveled off during that interval. It does not by itself establish equilibrium or adequate sampling. Inspect the trajectory alongside RMSF and radius of gyration to distinguish local loop motion from a broader rearrangement; compare independent runs when drawing conclusions about stability.

RMSF

Root mean square fluctuation per residue captures local flexibility. High RMSF values correspond to mobile loops, termini, and disordered regions. Low RMSF indicates rigid core residues or those involved in stable secondary structure elements.

Radius of gyration

Measures overall compactness. A stable radius of gyration indicates the protein maintains its fold. A sharp increase suggests partial unfolding or domain separation.

Secondary structure (DSSP)

Per-residue secondary structure assignment across all frames. Tracks helix-to-coil transitions, beta-sheet stability, and transient structure formation. The summary contains native counts for each secondary structure type per frame, rather than percentages. See the GROMACS DSSP reference for the assignment codes and output definitions.

Potential energy

Should decrease during minimization and stabilize during production. Large fluctuations during production may indicate simulation instability.

GROMACS energies are in kJ/mol. energy_log_native_units.csv and final_energy_kJ_per_mol use explicit native units. Older CSV column names and final_energy_kJ remain compatible aliases with the same numerical values; their _kJ suffix does not mean total energy in kilojoules. The original XVG and EDR files are retained in the archive.

Additional analyses

• Hydrogen bonds: Native GROMACS protein-protein hydrogen-bond indices and per-frame counts
• Ramachandran angles: Native GROMACS $\phi$/$\psi$ output
• SASA: Total and per-residue solvent-accessible surface area from gmx sasa
• Residue distance map: Mean minimum distance between residue pairs from gmx mdmat
• B-factors: Backbone fluctuation values written into a PDB by gmx rmsf

Limitations

• Protein preparation supports standard amino acid residues. Native TPR or native file modes support other systems when their required parameters are already supplied.
• Simulations can run on CPU or A10G GPU offload depending on system size, duration, and backend selection. A failed GPU run is reported and is not silently retried on CPU.
• The maximum simulation duration is 200 ns, which may be insufficient for slow conformational transitions, protein folding, or large-scale domain motions that occur on microsecond timescales.
• Protein preparation releases its setup restraints for production. Native systems retain the restraints and other settings encoded in the submitted inputs.
• Ligands, cofactors, covalent modifications, and custom residues require valid GROMACS parameters. This tool does not generate ligand topologies or custom force-field files.