This section is part of the GROMACS Wizard tutorial.
Once the system has been preprocessed and validated, we can start the preparation step. To launch GROMACS simulations, we first need to prepare the system:
- Choose the model (force field and solvent).
- Choose the molecular system and, optionally, choose a set of conformations or a path for a batch project.
- Define the periodic box.
- Choose what and how many ions should be added.
- Run preparation
During the preparation step, GROMACS Wizard will generate a model, and add solvent and ions if necessary.
Note: The Prepare step allows you to prepare either a single or a batch project for the same molecular structure based on a set of conformations or a trajectory that might be useful, e.g., for Umbrella Sampling. Below we first show how to prepare a single project, the batch project can be prepared in the same way by additionally specifying a set of conformations or a trajectory (path) corresponding to the system.
Choosing the results folder
First, let’s choose a folder where we want the results to be saved. Click on the … button to choose the folder.
The Open button allows you to open this folder in your default file explorer.
1. Specifying the model
The next step is to specify the model:
- Choose the force field.
- Optionally, provide additional topology files if your system contains molecules/residues not described by the force field (e.g., ligands).
- Choose the solvent model.
Choosing the force field
Now, we need to choose the force field with which we would like to simulate the system.
Please note, that the choice of the force field is important and might significantly influence the simulation. Please refer to publications on the force fields and select the most applicable to your system.
For this tutorial, we will be using the all-atom OPLS-AA/L force field. Please choose it from the available force fields in the Model section.
Note that the force field list contains standard force fields shipped with GROMACS and custom force fields you might have provided in GROMACS Wizard before. If you would like to use a custom force field, please refer to the Using a custom force field section below.
Providing additional topology files
The 1AKI system used in this tutorial has no arbitrary molecules so you do not need to provide any additional topology files.
If your system has any arbitrary molecules that you would like to include in the simulation and for which GROMACS cannot generate the topology itself, then you will need to provide the include topology files for them (.itp files). You can generate the topology files for arbitrary molecules using various servers, e.g. the ATB Server or CGenFF server. If you have them then you just need to provide them in GROMACS Wizard, and it will do the rest for you.
Please note that if you provide additional topology files then it is necessary to provide the force field with which they were parametrized. For example, if you generated ITP files using the ATB server then download the corresponding force field from this server (e.g., gromos54a7_atb). Please see the Using a custom force field section to see how to provide a custom force field.
Using a custom force field
If you want to use a custom force field then you can easily provide it by clicking on the “Add” 
button for force fields.
To remove an added force field from the list, click the “Remove” 
button. Please note, that it will remove all the added custom force fields, but the standard force fields will not be removed.
Choosing solvent
Each force field proposes water models that can be used to describe water molecules if you want to simulate your structure using explicit water.
In this tutorial, we will be using the SPC/E water model. Please choose the SPC/E model from the water model list:
Note that if you choose “none” as the water model then it won’t be possible to add solvent and ions.
To add explicit solvent in the system, check the Add solvent option as shown below:
Using a custom solvent model
If you want to use a solvent model that is not included with the chosen force field then you can easily provide it by clicking on the corresponding “Add” button for the water model. In the pop-up dialog, you can provide a topology file (.itp) for a new solvent model and, if necessary, a coordinate file (.gro), see the description below.
How to choose the solvent structure type
If the solvent is a 3-, 4-, or 5-site water model then you can choose the corresponding type from the list without the need to provide the corresponding structure file, in this case, the corresponding standard coordinate files shipped with GROMACS will be used.
Otherwise, you will need to provide a custom coordinate file (.gro) that corresponds to this solvent model. The coordinate structure file should contain a box of solvent molecules equilibrated in periodic boundary conditions to ensure a good alignment of molecules on the stacking interfaces. The box of solute is then built by stacking the coordinates read from the coordinate file.
To remove an added solvent model from the list, click the corresponding “Remove” button. Please note, that it will remove all the added custom solvent models from the chosen force field, but the standard water models provided with the force field will not be removed.
Existing hydrogens
If your system contains hydrogens you might choose to ignore them by checking the associated box in the Model section.
This is especially useful for NMR structures. Otherwise, if hydrogen atoms are present, they must be named exactly how the chosen force field expects them to be named (please check the chosen force field conventions). If you need to preserve hydrogens in the system as they are, then do not apply this option and make sure that all the hydrogens in the system are set and named properly.
We recommend ignoring existing hydrogens – GROMACS will then add them using the selected force field naming convention (note that not all the present force fields might have the same H atoms naming convention).
2. Choosing the system
Next, we need to specify the molecular system that we would like to prepare:
- Select the system in the Document View. If there are several structural models in the active document then select in the Document View the ones that you would like to prepare. If the active document contains only the system you would like to prepare then there is no need to select it – it will be selected automatically.
- Click the “Set system” button. If the selection was left empty and there are several structural models in the active document, it will ask you to choose whether you want to include all the structural models when preparing the system.
Once you set the system it should look like that:
Adding custom index groups
This is an optional step.
GROMACS will automatically generate standard index groups for your system based on its structure (e.g., protein, water, ions, etc). But you can add custom index groups that might later be useful for analysis or during the simulation (e.g., as pull coordinate groups). This allows you to use the extensive selection mechanisms available in SAMSON (see User Guide: Selecting).
Requirement: This step requires that the system has unique and consecutive indices for residues and atoms.
Note: The new index groups are added using gmx make_ndx command and are saved in the “index.ndx” file in the project folder.
Note: You can add new index groups in the next steps (minimization, equilibration, simulation).
To add a custom index group click the “Add/edit index groups” button.
This will open a pop-up window in which you can specify the new index groups.
To specify new groups, you can use either the GROMACS selection syntax or the extensive selection mechanisms available in SAMSON (see User Guide: Selecting). In the latter case, the GROMACS selection string will be automatically generated for you based on your selection in SAMSON.
Let’s see an example: we will add an index group for residues that are in alpha helices.
First, we will perform the selection in SAMSON using Select menu > Residues > Amino acids > Secondary structure > Alpha helices.
Then in the Index Groups window click the “Generate based on current selection in document” button.
This will automatically generate a GROMACS selection string using residue or atom selection syntaxes. Name the new group, e.g. “HELICES“. You can also test the selection string and select nodes in SAMSON based on it to test it. To register the new index group click the “Add index group to the list” button.
The new group will be added to the list:
Batch preparation
This is an optional step if you want to prepare a batch project for a system based on a set of conformations or a trajectory. This can be useful, for example, for Umbrella Sampling. The batch project allows you to easily launch the next steps (minimization, equilibration, simulation) for the whole batch (locally or in the cloud) and not per project.
To set up the batch project:
- Check the “Batch preparation” box.
- Select in the Document view a set of conformations or a single trajectory (a path node) corresponding to the chosen system.
- Click the “Set conformations or a path” button.
Please note that for the batch project, the periodic box is set either based on all the conformations (option: “Box lengths”) or per conformation (option: “Solute-box distance”). See the next section for more information.
Note: The prepared batch project will contain numbered subfolders each representing a fully separate project. You can run the next steps on the whole batch project or separate subfolders.
3. Defining the box
Now we need to define the box before proceeding with the preparation step.
To define the box, select the system from the Document view or leave the selection empty if you would like to prepare the whole system in your document.
Several types of unit cells are supported:
- Cubic,
- Orthorhombic,
- Triclinic,
- Rhombic dodecahedron,
- Truncated octahedron.
Please refer to GROMACS Wizard: Periodic boundary conditions for more information.
Two options for initial fitting of the box based on the system are available:
- Box lengths – specify the box size. On fitting it will fit the box tightly to your system – you will need to increase the size as needed to ensure the minimum image convention. This option also allows you to move the box to position the system in it. For the batch project, the initial box size is determined based on all the conformations or the whole path – choose this option if you want the box size to be the same for the whole batch.
- Solute-box distance – specify the distance between the system (solute) and the box. At least 1 nm is recommended to ensure the minimum image convention. For the batch project, the box sizes will be different per each conformation or frame of the path.
For the sake of the tutorial, choose the Orthorhombic unit cell, then click “Compute fitted box“, and choose the “Solute-box distance” option, as shown below.
This will automatically generate the chosen box (its positions and size) for your system. All the atoms of your selected system will be included in the box because the box dimensions are determined based on the size of your system (in x, y, z).
Once you have generated your box, you can easily change its size by modifying the corresponding unit cell parameters.
Note: the box has to include all the structures selected for the preparation.
In the case of the “Box lengths” option, it is necessary to increase the box size to add enough additional space between the system (without solvent) and the box edges. Since the periodic boundary conditions are used it is crucial to satisfy the minimum image convention. This means that, for example, a protein should never see its periodic image, otherwise the calculated forces might be wrong. We recommend adding a distance of at least 1.0 nm – that would mean that there is at least 2.0 nm between any two periodic images of a protein or other non-solvent molecules (please note that molecules in the system, e.g. a protein, might go through various conformations). This distance should be sufficient for just about any cutoff scheme commonly used in simulations but you might need to verify with the force field paper if you are using a custom force field.
Note: When loading GROMACS results, SAMSON will try to automatically detect the type of the unit cell of the system, but you can always modify it in the importer dialog which appears on loading of GROMACS trajectories.
4. Neutralizing the system
We need to make sure the system is neutral (has zero charge) by adding positive or negative ions.
Note: ions are added to the system as a replacement for water molecules, so solvation is required if you want to add ions.
To neutralize the system, check the Neutralize system option as shown below:
The neutralization will be done automatically by GROMACS.
Each force field proposes a set of positive and negative ions for the neutralization of your system by GROMACS. You can choose them from the associated lists. Note that only one type of ions (positive or negative) or none of them will be added by GROMACS based on the total charge of your system.
If you would like to know the total charge of your system before running the preparation step, you can compute it by selecting the system and pressing the Update 
button. For example, if your system’s total charge is 8, GROMACS will add 8 negative ions to neutralize the system.
Additional ions
You can increase the ion concentration in your system by adding more ions. To do so, check the “Add additional ions” box. Two options are available:
- Number of ions – specify the number of additional positive and negative ions that you would like to be added to the system. For example, if your system’s total charge is 8, you can ask GROMACS to add 12 additional positive ions thus bearing the total charge of the system to 20. Since GROMACS needs to neutralize the system it will add 20 negative ions during the preparation stage. Therefore, the ions that would be added to your system to neutralize its total charge go from 8 negative ions to 20 negative ions and 12 positive ions.
- Salt concentration – specify salt concentration (mol/liter). This will add sufficient ions to reach up to the specified concentration as computed from the volume of the cell in the input .tpr file. If you specify a salt concentration existing ions are not taken into account. In effect, you therefore specify the amount of salt to be added on top of neutralizing conterions. This will add sufficient ions to reach up to the specified concentration as computed from the volume of the cell. These ions will be added on top of neutralizing counterions to reach up to the specified concentration as computed from the volume of the cell.
Note: ions are added to the system as a replacement for water molecules.
Run preparation
You can remove the initial structure by checking the “Remove initial structure” option.
Now it should be all set to prepare the system – click the Prepare button to run the preparation step.
Some pop-ups might appear informing you about the current steps or possible warnings/issues if there are any.
To view the log/output click on the Output button at the top of the GROMACS Wizard:
Results
Once the preparation is finished, an import dialog will appear asking whether you want to import the prepared system into SAMSON.
If you choose to load it, a new structural model representing the prepared system should be visible in the Document view and the Viewport.
A folder with all the available index groups (including custom index groups) will be created. Double-click on any of them to select the corresponding nodes.
Visualization
If you left the “Show proteins as ribbons” option checked in the importer then it should automatically apply visualization for the protein in your system: add the Ribbons visual model and hide protein residues.
You can apply more visualizations via the Visualization menu, for example via Visualization menu > Visual model > Visual preset.
Please check out the User Guide: Visualizing to learn more about how to add visual models, change rendering, and create publication-quality images.
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