A reaction-coordinate trajectory can show how two molecular groups move relative to one another, but it is not yet a potential of mean force (PMF). To estimate a PMF with umbrella sampling, you need a set of starting conformations spread along the coordinate, followed by simulations that sample each window. Turning a trajectory into that organized set—and keeping the coordinate consistent throughout the workflow—is a common source of confusion.
This guide focuses on that handoff: using GROMACS Wizard to select umbrella windows from a GROMACS trajectory, then setting up the simulations that produce data for PMF analysis.
First, choose where your window conformations come from
There are two routes. If you already have a GROMACS trajectory, you can select frames from it as the initial conformations for the windows. For example, the trajectory may come from a COM pulling simulation. If your conformations or pathway already exist in SAMSON, use those as the starting states instead; the batch-computations workflow can be followed up to NVT equilibration.
This distinction matters because the starting conformations determine how you create the batch project. In the trajectory route, GROMACS Wizard can identify the trajectory in the selected input project. In the existing-conformations route, you begin from the batch of states you already have. In either case, the later NPT equilibration and production simulations need the umbrella-specific COM pulling parameters for the coordinate you plan to analyze.
Choose windows along the coordinate—not just along time
In the Umbrella Sampling tab, select the input project and define the reaction coordinate by choosing two index groups. The coordinate plot shows distance versus time and marks suggested initial conformations. Check that the groups describe the molecular separation you actually want to study; the windows and the later analysis depend on that choice.
There are two ways to control how starting conformations are selected:
- Number of conformations: choose a total number, and the conformations are distributed along the reaction coordinate.
- Minimum COM spacing: specify a minimum center-of-mass distance between selected conformations.
The first option is useful when you have a target number of windows in mind. The second is useful when you want selection to respect a distance interval. You can also limit the selection to a start and end frame. That gives you a way to focus on the portion of the trajectory relevant to your question rather than automatically using its full duration.
When you click Generate project, GROMACS Wizard creates a timestamped batch-project folder with the _umbrella suffix. Each subfolder represents one umbrella window. The frames.ndx file records which frames from the original trajectory were selected—a useful record when you need to check where the starting structures came from.
Keep the coordinate, but stop the pulling
For each window, run NPT equilibration and then production MD with the COM pulling parameters for the reaction coordinate. A key detail is that these parameters must also be present during NPT equilibration because they affect the system’s behavior. For the tutorial’s example, the coordinate is the distance between the centers of mass of chains A and B:
- Type:
umbrella; geometry:distance. - Groups: chain A and chain B.
- Start:
yes; distance components:N N Y(the z-direction). - Initial value:
0 nm; force constant:1000 kJ mol^-1 nm^-2. - Rate:
0 nm/ps.
The zero rate is important: it means the coordinate is not being actively pulled during these simulations. The umbrella setup is used for sampling at the selected window, rather than continuing the motion that may have been used to generate the path.
Run the batch, then analyze the result
Choose the batch project as the input for NPT equilibration and production MD. The auto-fill control can populate the input path from the previous step. When running locally, a batch project is handled as separate jobs—one per window—visible in the Local jobs window. Once the umbrella simulations are complete, continue to PMF analysis, where the window data can be used to compute the PMF with WHAM.
A simple workflow check is to confirm three things before launching: the chosen index groups match the intended reaction coordinate; the selected frames cover the region you want to analyze; and the COM pulling coordinate is consistent across the windows, with the rate set to zero for equilibration and production. These checks help prevent avoidable mismatches between the pathway, the simulations, and the analysis.
For the full walkthrough, see the GROMACS Wizard Umbrella Sampling tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.