Suggested workflow for GROMACS simulations

This part of the GROMACS best practice guide walks you through a suggested workflow for preparing, running, and analysing the results of your GROMACS simulations. There are already many very good tutorials available to teach new users how to do this (see below). Here, we have collated and compiled the suggestions of these tutorials into what we believe to be the best way to prepare and run a GROMACS simulation.

This section is divided as follows: first we will describe how to choose and prepare the input files for your system; we will then discuss the correct way to run your GROMACS simulations; and we will finish by discussing some of the post-analysis tools that are available.

Note that this section is written with the intent of helping users to use GROMACS. Some of the sections use examples of common shell-scripting practices – it is assumed that the reader is either familiar with basic shell-scripting or knows where to learn more about this. Likewise, the background theory behind some of the techniques discussed in the post-analysis section is not presented here. Where possible, we have added links to places where this theory is explained.

Preparing the system

PDB2GMX

The GROMACS pdb2gmx command is used to convert a coordinate file into a set of GROMACS topology files (in these examples, we will assume that the file is a .pdb file, but this is not a necessity). To run this:

gmx pdb2gmx -f ${INPUT_FILE}.pdb

where ${INPUT_FILE}.pdb is replaced with the input file name. You will be prompted to select the forcefield you would like to use. GROMACS comes with a number of AMBER and GROMOS forcefields, as well as a CHARMM and an OPLS-AA option. You will also need to specify your water model (choices included are TIP models, and the SPC and SPC/E models). Specifying the water model here results in pdb2gmx to write a complete topology and will ensure that all topology files are consistent if the system needs to be hydrated. The code above produces three outputs: a system topology topol.top, a position restraint file posre.itp (included in the topology file), and a coordinate file conf.gro. Further to these files, pdb2gmx will output a number of interesting details to screen, such as the total mass of the system given the coordinates and topology being used as well as the net charge of the system. The charge is particularly important to note down and will be used in the `Solvating and ionise a system`_ step of system preparation.

More information about the flags and options of this program can be found in the GROMACS `PDB2GMX manual<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-pdb2gmx.html>`_.

Generating your own forcefield file

Note

It is advised to use one of the pre-existing GROMACS forcefields where possible. Only consider generating your own forcefield if the ones available on GROMACS do not fulfill your requirements.

GROMACS comes with a number of forcefields available out of the box. These forcefields are stored within the main GROMACS directory in share/gromacs/top. If the forcefield you want to use is not present, you will need to generate your own forcefield files. To do this, create a directory <forcefield>.ff in your working directory, with <forcefield> replaced by a sensible name. Within this directory, create a forcefield.doc file and write a simple one-sentence description of your forcefield – this description is what will come up in pdb2gmx when you choose a forcefield. Next, generate a forcefield.itp included topology file. This file is a topology file where you can define the parameters for atoms, bond, angles, dihedrals, etc.. You can find more information about generating topology files from scratch in the GROMACS manual `file format page<http://manual.gromacs.org/documentation/current/reference-manual/file-formats.html#top>`_.

Using a <forcefield>.ff directory has a number of advantages over writing out your system topologies directly. For one, this allows for better reproducibility in the even that you want to simulate a new system with this forcefield. It also has a number of functionalities that can be useful. For instance, adding a watermodels.dat file into the forcefield directory makes it easy to keep track of water models available. A line and descripting can be added in this file for each water-model included topology file. This file is what prompts the choice of water model in pdb2gmx.

Once it is populated, running pdb2gmx in the directory containing your <forcefield>.ff directory will result in your new forcefield being included at the top of the list of selectable forcefields. If you are happy with your <forcefield>.ff directory and you will use it a lot (and if you have the correct permissions to edit parts of the GROMACS directory), you can copy it to the share/gromacs/top of the GROMACS directory (or to $GMXLIB which should be the same directory). In doing so, your forcefield will become a permanent part of the forcefields that pdb2gmx can use.

Note

You can also generate an Amber or CHARMM topology by using the AmberTool antechamber function or the CHARMM cgenff function. To do this, you should follow the procedures described above, making sure to select an appropriate forcefield from the selection GROMACS provides. Then, use a parameter-generating tool like antechamber with actype (for Amber) or cgenff (for CHARMM). The topologies generated in this way can then be added to the GROMACS topology that you generated. This can be done by opening the GROMACS topology file and including the following line at the start:

#include "/path/to/forcefield_file.itp"

where the path is to the topology file generated in antechamber or cgenff.

For more information on generating your own forcefield, please see the GROMACS manual pages about `adding a residue<http://manual.gromacs.org/documentation/current/how-to/topology.html>`_ and `force field organisations<http://manual.gromacs.org/documentation/current/reference-manual/topologies/force-field-organization.html>`_.

Preparing and solvating your simulation box

Generating a system of replicates from a GROMACS structure file

It is possible to populate a simulation box with molecules by replicating the contents of a GROMACS structure file (.gro) multiple times. This can be achieved with the insert-molecules command. While any structure file can be used (including crowded system file), this is particularly useful if you want to create a system with a large number of copies of a single molecule (i.e. as found in a lipid bilayer or a non-aqueous solvent). Furthermore, the topology (.top) file generated for the system to be replicated will still work for the new, larger system, by including the total number of molecules in the directive [molecules].

To generate a system using this command, run:

gmx insert-molecules -ci ${INPUT}.gro -o ${OUTPUT}.gro \
                     -nmol ${N} -box ${X_LENGTH} ${Y_LENGTH} ${Z_LENGTH}

where ${INPUT}.gro is the structure file of the molecule/system you wish to replicate, ${OUTPUT}.gro is the output file, ${N} is the number of times that the contents of ${INPUT}.gro will be replicated, and ${X_LENGTH}, ${Y_LENGTH}, and ${Z_LENGTH} are the dimensions of the cubic box into which these ${N} replicas must be packed.

There are number of further options to help pack your system, including a way of defining the default van der Waals distance between atoms in your system, a way of inserting new molecules into an existing system, and methods to control the amount of random rotation that replicated molecules can undergo. All of these options can be found in the `gmx insert-molecules<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-insert-molecules.html>`_ page of the GROMACS manual.

Generating a simulation box

Now that a topology has been generated, the next step is to generate a simulation box into which to place this topology. For this, use the editconf command. This tool has a number of functionalities, including generating and orienting a simulation box, and filing it with pre-generated topologies. To create a simulation box with editconf, run:

gmx editconf -f ${INPUT}.gro -center -d ${SEPARATION} -bt ${BOX_TYPE} \
             -o ${OUTPUT}.gro

where ${INPUT}.gro is the input forcefield-compliant coordinate file, ${OUTPUT}.gro is the chosen output name (the default is out.gro), the -c flag will place the system described in ${INPUT}.gro into the centre of the simulation box, -d ${SEPARATION} defines the minimum separation between the input and the edge of the box (units are in nm), and -bt ${BOX_TYPE} defines the type of box for the simulation (triclinic is the default, but other options are cubic, octohedral, or dodecahedral). There are a number of other editconf options, predominantly to have more control over defining the simulation box. These can be found in the GROMACS manual `gmx editconf page<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-editconf.html>`_.

Solvating a system

The aptly-named solvate tool can be used to create a box of solvent or to solvate a pre-existing box. To use it, run:

gmx solvate -cp ${SOLUTE}.gro -cs ${SOLVENT}.gro -p ${TOPOLOGY}.top \
            -o ${OUTPUT}.gro

where ${SOLUTE}.gro is the simulation box configured using the steps described above, ${SOLVENT}.gro is the solvent configuration file (note that GROMACS has a number of pre-defined solvent configuration files but that you can also prepare and use your own), and ${TOPOLOGY}.top is the topology obtained when running `GMX2PDB`_. If using a GROMACS-provided solvent, the addition of this solvent should not alter the net charge of the system.

For further information, please see the GROMACS manual `gmx solvate<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-solvate.html>`_

Adding ions and creating a charge-neutral system

Adding ions to your solvated system can serve two purposes: it can help to neutralise any charge in your system; and it allows you to simulate systems with similar salt concentrations to their real-world equivalents. Adding ions is done in two parts: first, you need to use the grompp tool to generate a .tpr file to be used when adding ions, and then you must replace some of the recently-added solvent molecules with the necessary counterions using genion.

The GROMACS preprocessor tool grompp reads in coordinate and topology files to generate an atomic-level input file (with a .tpr extension). This .tpr file contains all of the parameters needed for all atoms in the system. We will go into more details about the grompp tool in the Running a simulation section. For now, the important part is that, to generate a run input .tpr file, grompp needs a structure (.gro) file, a topology (.top) file, and a file defining the instructions for the simulation run (this is kept in an .mdp file). This .mdp file can be kept empty when ionising the system as no actual simulation is to be run. To generate the ,tpr file, run:

gmx grompp -f ${RUN_FILE}.mdp -c ${COORDINATES}.gro -p ${TOPOLOGY}.top \
           -o ${OUTPUT}.tpr

It is likely that grompp will output a number of notes to screen (one of which should be reminding you of the net non-zero charge of your system). In this case, these can be ignored (this is an exception and is not usually true).

Now that the .tpr has been generated, genion can be used to make the charge of the system neutral. The system charge is decreased by replacing a number of parts of the system with anions and cations. This is done by running the following (note that the ${INPUT}.tpr named below is likely to be the ${OUTPUT.tpr} generated in the grompp step above):

gmx genion -s {INPUT}.tpr -p ${TOPOLOGY}.top -neutral -o ${OUTPUT}.gro

You will be prompted to choose the group within your system (solvents, solutes, protein backbones, etc.) that you would like ions to replace, with the frequency of occurrence of each group also shown. Note that some groups may have overlap completely and be different names for the same group. In general, it is best to replace solvent molecules with ions (the group named SOL). Once a group is chosen, genion will replace a number of that group with anions and cations until the system is charge neutral. The default anion name is CL, though this name can be changed with the -nname flag, and the default cation name is NA`, but this name can be changed with the ``nname flag. By default, the cation and anion charges are 1 and -1 respectively, but this can be changed with the -pq flag for the cation and the -nq flag for the anion.

For further information, please see the GROMACS manual `gmx grompp<http://manual.gromacs.org/current/onlinehelp/gmx-grompp.html>`_, and `gmx genion<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-genion.html>`_ pages.

Running a simulation

This section describes how to set the GROMACS simulation parameters, how to generate a run input file from a GROMACS topology and parameter file, how to run a simulation in GROMACS, and how to analyse the results produced. It is assumed that you already have a system topology ready to use (by following the steps in the Preparing the system section) – if this is not the case, and if you are unsure how to create this topology, please read through that section.

Creating a run parameter file

A GROMACS molecular dynamics parameter (.mdp) file defines the simulation parameters to be used during a simulation. A number of options can be set in this script, including: defining the simulation integrator that will define the method used to solve Newton’s equations to propagate the system forward in time; setting the size of the simulation timestep and total simulation time; setting the restrictions within which the system will be simulated (such as setting a system pressure/temperature through a thermostat or barostat); setting or adjusting the way the simulation forcefield is interpreted (by e.g. defining the way short- and long-ranged interactions are calculated and at what distance they are truncated); to define which simulation properties to output (and the output frequency); and many more options. Given the number of options and variables that can be included, not included, or kept as default, we will not go over all of the options here and will instead look at and explain an example molecular dynamics parameter file. You can find a list of all available options in the GROMACS manual `molecular dynamics parameters page<http://manual.gromacs.org/documentation/current/user-guide/mdp-options.html>`_.

Example molecular dynamics parameter file

Note

GROMACS has been developed to be forcefield agnostic. This means that a large number of different forcefields can be run using GROMACS. However, this also means that different forcefields will require slightly different constraints to be defined in their “.mdp” parameter files. You can find more about this in the `Force fields in GROMACS<http://manual.gromacs.org/documentation/current/user-guide/force-fields.html>`_ section of the GROMACS manual.

The GROMACS manual has the following `example script<http://manual.gromacs.org/documentation/current/user-guide/file-formats.html#mdp>`_:

; Intergrator, timestep, and total run time
integrator               = md
dt                       = 0.002
nsteps                   = 500000

; Logs and outputs
nstlog                   = 5000
nstenergy                = 5000

; Bond constraints
constraints              = all-bonds
constraint-algorithm     = lincs

; Van der Waals interactions
vdwtype                  = Cut-off
rvdw                     = 1.0
cutoff-scheme            = Verlet
DispCorr                 = EnerPres

; Coulombic interactions
coulombtype              = PME
rcoulomb                 = 1.0

; Thermostat
tcoupl                   = V-rescale
tc-grps                  = Protein  SOL
ref-t                    = 300      300

; Barostat
pcoupl                   = Parrinello-Rahman
ref-p                    = 1.0
tau-p                    = 2.0
compressibility          = 4.5e-5

Note

The parameters chosen for time step, bond constraints and van der Waals/electrostatic interactions depend on the force field being used.

First note that, while the the example above is ordered in a sensible way, with commands grouped by what they are defining (e.g. temperature, pressure, van der Waals interactions, etc.), the order in which the individual commands are written does not matter. Having said that, we would recommend grouping commands affecting similar simulation aspects together to help future readability. Also, if the same command appears twice in a .mdp file, the second appearance will override the first.

The first block of the example script defines the molecular dynamics integrator as a Verlet leap-frog algorithm (integrator = md), declares that the simulation timestep will be 2 fs (dt = 0.002, where the default unit is ps), and that the simulation will run for a total of 500,000 dt timesteps (nstep = 500000) or 1 ns.

The next block defines the simulation outputs. nstlog sets the time interval between each output to log (md.log) of the energy components and physical properties of the system at 5,000 dt. nstenergy sets the time interval between each output to the energy file (ener.edr) of the energy components of the system at 5,000 dt – note that this file is written in binary.

In this example, all bonds are constrained and set to be rigid. This is done with the constraints = all-bonds command. Furthermore, the constraint algorithm is set to be the linear constraint solver algorithm with the constraint-algortihm = LINCS command.

The van der Waals interactions are set as truncated (vdwtype = cutoff), with a cutoff distance of 1 nm (rvdw = 1.0). This means that no van der Waals interactions will be computed for pairs of particles whose centre-of-mass separation greater than 1 nm. To save in simulation time, a neighbour-list cutoff scheme is used. The cutoff-scheme = Verlet command specifies how this list is generated. A long-ranged dispersion correction to the energy and pressure is considered here with the DispCorr = EnerPress command. The Coulombic interactions will be calculated using the smooth particle-mesh Ewald (SPME) method (coulombtype = PME), with an interaction cutoff of 1 nm (rcoulomb = 1.0).

The thermostat used for this simulation is defined by the tcoupl = v-rescale – in this case, the velocity rescaling algorithm is used. The tc-grps is there to specify that the protein and solvent (SOL) should have separate heat baths for this simulations. The reference temperature (or desired temperature) is set by ref-t. In this case, the reference temperature for both the protein and the solvent have been set to 300 K. Note that the reference temperature must be set for every group defined in tc-grps and that these temperatures do not need to be the same.

In this example script, the barostat is defined with the pcoul parameter as the Parinello-Rahman barostat. The reference (or desired) pressure is set at 1 atm with the ref-p command, and the coupling time constant tau-p is set to 2 ps. Much like the temperature coupling time constant set for the thermostat, the pressure coupling time constant is used to dictate the frequency and amplitude of fluctuations during a simulation. Finally, the compressibility parameter is used to define the compressibility of the system (how the volume of the system changes as pressure is changed). In this case, it is set as 4.5e-5 bar^-1 (water compressibility).

Generating your simulation input file

Once you have prepared your .mdp file, you are ready to combine it with the topology you’ve prepared to create a run input .tpr file. For this, we will use the GROMACS pre-processing tool grompp. This is very similar to the step described in the `Creating a charge-neutral system`_ section, but with more care regarding the warnings that are output. Like before, this is done by running:

gmx grompp -f ${RUN_FILE}.mdp -c ${COORDINATES}.gro -p ${TOPOLOGY}.top \
           -o ${OUTPUT}.tpr

where ${RUN_FILE}.mdp is file discussed in Creating a run parameter file, and ${COORDINATES}.gro and ${TOPOLOGY}.top were generated following the instructions in the Preparing the system section. The ${OUTPUT}.tpr file generated here is the only file needed to proceed with running a GROMACS molecular dynamics simulation.

Running your simulation

With the run input .tpr file now generated, we are ready to run a GROMACS simulation. For this, we will use the mdrun command:

gmx mdrun -s ${INPUT}.tpr

This command will run the simulation with the topology that you’ve prepared and the molecular dynamics parameters that you’ve chosen.

Once the simulation is complete, mdrun will have produced a number of files. The ener.edr file is a semi-binary file that contains all of the thermodynamic information output during the run (e.g. energy breakdowns, instantaneous presssure and temperature, system denstity, etc.). Likewise, the md.log file generated outputs these properties, but in a text format. The traj.trr file is a binary that contains details of the simulation trajectory and xtc file is a compressed and portable format for trajectories. The final file produced by default is the counfout.gro is a text file containing the particle coordinates and velocities for the final step of the simulation.

It is possible to add flags to mdrun to alter some of the parameters that had been set in the molecular dynamics parameter file. For instance, the -nsteps flag can be used to override the number of timesteps that the simulation should run for. Also, there are a number of useful options for defining input files (and input file types), output files, and parameters related to the computational system on which you are running (such as the -nt option to set the number of MPI threads that the simulation should use). More information on these and other options can be found on the GROMACS `gmx mdrun<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-mdrun.html>`_ page.

Post-processing and analysis tools

With the simulation complete, we can analyse the simulation trajectory and understand what the simulation has demonstrated. GROMACS offers a number of post-simulation analysis tools. In this section, we will discuss tools that can be used to: generate the thermodynamic properties of interest; obtain radial distribution functions and correlation functions;

Thermodynamic properties of the system

The GROMACS energy tool can be used to extract energy components from an energy (.edr) file. By default, this tool will generate an XMGrace file. To use this, run:

gmx energy -f ${INPUT_ENERGY_FILE}.edr -o ${OUTPUT_XMGRACE_FILE}.xvg

When running this, you will get a prompt asking which property you would like output (e.g. potential energy, kinetic energy, pressure, temperature, etc.). Enter the correct number to generate an XMGrace file that, when plotted, will show you how that property varied over the simulation run. There are a number of other options for the energy command, and these can be found in the GROMACS manual `gmx energy<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-energy.html#gmx-.. energy>`_ page.

Generating an index file

GROMACS has a post-analysis tool for generating radial distribution functions (RDFs). Before generating an RDF, we will need to create a GROMACS index (.ndx) file to categorise the various parts that compose the simulation into indexed groups. This can be done with the gmx make_ndx command. To use it, run:

gmx make_ndx -f ${INPUT}.gro -o ${OUTPUT}.ndx

where ${INPUT}.gro is a GROMACS configuration file for the trajectory you are wanting to calculate the RDF for. Provided you used the default names in your mdrun, you can simply use confout.gro. The make_ndx command will analyse the system, and output the default index groups. It is possible to create new index groups by using the command prompts listed (for instance, you can create a group composed of only the oxygens from the solvent waters by running a OW within make_ndx). For more information, please see the GROMACS manual `gmx make_ndx<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-make_ndx.html>`_ page.

For more complex manipulations than selecting all of one group of atoms, GROMACS provides the gmx select option. This will allow you to define the exact time or particles or regions of interest within your simulation. You can find more information on how to use this in the GROMACS manual `Groups and Selections<https://manual.gromacs.org/documentation/2019/reference-manual/analysis/using-groups.html#selections>`_ page.

Radial distribution function

Once an appropriate index file is generated, with the atoms for which an RDF is to be calculated indexed into appropriate groups, we can use the gmx rdf command to generate the RDFs. This is done by running:

gmx rdf -f ${TRAJECTORY_INPUT}.trr -n ${INDEX_INPUT}.ndx  \
        -ref ${REFERENCE_GROUP} -sel ${SELECTED_GROUP} -bin ${BIN_WIDTH}
        -o ${OUTPUT}.xvg

where ${TRAJECTORY_INPUT}.trr is the trajectory file for which you would like to generate an RDF, and ${INDEX_INPUT}.ndx is the index file that you produced using make_ndx. ${REFERENCE_GROUP} should be replaced with the name of the principal group to be used in the RDF as it appears in the ${INDEX_INPUT}.ndx file. Likewise, ${SELECTED_GROUP} should be replaced with the name of the atom group(s) for which you want to calculate the RDF against the position of the reference group (e.g. if you want to calculate the RDF between sodium ions and chloride ions, your reference group would be one of NA or CL, and your selected group would be the one not chosen as reference). Note that it is possible for your reference and selected groups to be the same group.

Mean squared displacement and velocity autocorrelation functions

Gromacs offers a number of tools to calculate correlation and autocorrelation functions. Here, we will look at two specific example: the mean-squared displacement (MSD) and velocity autocorrelation function (VACF). We will focus on how to generate these functions within GROMACS but you can use these links to find an overview of the theory behind the `MSD<http://manual.gromacs.org/documentation/current/reference-manual/analysis/mean-square-displacement.html>`_ and the `VACF<http://manual.gromacs.org/documentation/2019/reference-manual/analysis/correlation-function.html>`_.

Calculating the MSD of parts of a system can be done using the gmx msd. This can be run using:

gmx msd -f ${INPUT_TRAJECTORY}.trr -s ${INPUT_TOPOLOGY}.tpr -o ${OUTPUT}.xvg

where ${INPUT_TRAJECTORY}.trr is the trajectory file of the simulation for which the MSD is being calculated, and ${INPUT_TOPOLOGY}.tpr can be the input file used to obtain this trajectory (note that it is possible to use the final topology confout.gro file here instead to obtain the same results). Running this command will prompt you to choose the group for which you would like the MSD. Note that, if the group you are looking for is not present in the list, you can generate an index file (see Generating an index file) where you can define this new group. To include this index file, add the option -n ${INDEX_FILE}.ndx to the command above. For more information and options, please look at the GROMACS manual page on the `gmx msd command<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-msd.html#gmx-msd>`_.

VACFs can be generated using the gmx velacc command:

gmx velacc -f ${INPUT_TRAJECTORY}.trr -o ${OUTPUT}.xvg

where ${INPUT_TRAJECTORY}.trr is the trajectory file of the simulation for which the VACF is being produced. You will get a prompt asking for which group of atoms the VACF should be calculated. If the group you want is not present, you may need to create it by following the instructions in the Generating an index file section of the manual. To include your index file, add it with the -n ${INPUT_INDEX}.ndx option. You can find more options and information on the GROMACS manual `gmx velacc<http://manual.gromacs.org/documentation/current/onlinehelp/gmx-velacc.html#gmx-velacc>`_ page.

Further resources

There are a number of excellent GROMACS tutorials that name a number of commands not mentioned here. The following tutorials are highly recommended:

Furthermore, the `GROMACS How-To guides<>http://manual.gromacs.org/documentation/current/how-to/index.html`_ provide a lot of information as well.