Umbrella sampling can produce many windowed simulations, but turning those separate pieces into a useful picture is a common sticking point. A Potential of Mean Force (PMF) brings the data together along a reaction coordinate. Yet a smooth-looking curve is not the whole story: you also need to know whether the coordinate was adequately sampled.
In GROMACS Wizard, the PMF is computed with the Weighted Histogram Analysis Method (WHAM). The analysis produces two complementary plots: the PMF profile and a histogram showing how the reaction-coordinate space is covered. Reading them together helps you understand both the estimated profile and where the underlying sampling may need attention.
Before computing: make sure the windows belong together
Start with the project generated by an umbrella-sampling workflow. The input folder should contain numbered subfolders that match the same molecular system and reaction-coordinate definition. This consistency matters: the analysis combines data across windows, so mixing projects or coordinate setups can make the result difficult to interpret.
In the WHAM Analysis tab of GROMACS Wizard, choose the project path. If the previous project you ran was the umbrella-sampling simulation, you can use auto-fill to populate the path. Then confirm that the folder contains the expected numbered subprojects.
Compute the profile
GROMACS Wizard loads information from the project, including the reaction-coordinate information, time range, and temperature. Choose the reaction coordinate you want to analyze. If appropriate for your analysis, you can adjust optional settings such as custom bounds, a custom time range, or energy units. Then click Compute.
For large trajectories, allow several seconds or minutes for the calculation. When it finishes, the tool generates the PMF profile and a histogram of coordinate coverage.
Use the histogram as a sampling check
The PMF is the result most people look for first, but the histogram is essential context. It shows how well the reaction-coordinate space is covered by the input data. Look for regions that appear sparsely represented: they can indicate that the sampling is not as strong there as elsewhere. Treat this as a prompt to investigate, rather than assuming that the profile alone tells you whether sampling is sufficient.
A practical review is to ask three questions:
- Is this the intended coordinate? Confirm that the selected reaction coordinate matches the umbrella-sampling setup.
- Does the coverage look adequate across the range? Use the histogram to identify regions that may need additional sampling.
- Are the analysis settings appropriate? Check any custom bounds, time range, or energy units you selected.
If some regions appear under-sampled, return to the umbrella-sampling workflow and add or extend simulations for the relevant windows. Then run the analysis again. This makes the histogram useful not just as a companion plot, but as a guide for deciding what to inspect next.
Keep results organized
GROMACS Wizard saves the profiles, histograms, and plots in the project’s wham_results subfolder. If you later switch reaction coordinates, the tool reuses already computed data for the same parameters. Keeping the analysis with its project makes it easier to revisit the results and understand which inputs produced them.
For the complete workflow and interface details, see the GROMACS Wizard PMF Analysis tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.