Umbrella sampling can produce a collection of simulations that are individually useful but difficult to interpret as a whole. The practical question is what those windows say together about the free-energy landscape—and whether they cover the reaction coordinate well enough to make the resulting profile useful.
In GROMACS Wizard, the Weighted Histogram Analysis Method (WHAM) combines umbrella-sampling data to calculate a Potential of Mean Force (PMF). The PMF profile is only part of the result: the accompanying histogram helps you inspect how the sampled windows cover the coordinate. Looking at both is a useful way to decide whether to proceed with interpretation or revisit the sampling.
Start with a consistent set of windows
Before opening WHAM Analysis, finish the umbrella-sampling workflow. The input folder should contain numbered subfolders that belong to the same system and use the same reaction-coordinate definition. This consistency matters: the analysis needs to combine related windows, not a mixture of projects or coordinate setups.
If you have just run umbrella sampling, use the auto-fill option in the WHAM Analysis tab to select the previous project path. Otherwise, choose the project folder yourself, then check that its subfolders match the intended system and coordinate. This small check can save time by catching an incorrect input path before computation begins.
Compute the PMF
Open the GROMACS Wizard and switch to the WHAM Analysis tab. After selecting the project, choose the reaction coordinate you want to analyze. GROMACS Wizard loads the reaction-coordinate information, time range, and temperature from the project automatically.
You can adjust optional settings, including custom bounds, a custom time range, or energy units, when your analysis calls for them. If you do not need to change those settings, avoid changing them simply for the sake of it: a clear record of the parameters used makes it easier to compare results later. When the inputs are ready, click Compute. For large trajectories, allow several seconds or minutes for the calculation.
Read the plots together
The output includes two plots: the PMF profile and a histogram showing coverage along the reaction coordinate. The PMF is the free-energy profile calculated from the windowed data. The histogram provides a practical view of where the coordinate space has data and where coverage may be sparse.
Do not treat a smooth-looking PMF curve by itself as proof that sampling was sufficient. Inspect the histogram alongside the profile, and note regions where coverage appears limited. Those regions are candidates for additional sampling; the plots help guide that decision, but they do not replace scientific judgment about whether the sampling is adequate for your question.
Use the result as part of an iteration
When coverage looks insufficient in part of the coordinate range, return to umbrella sampling and add or extend simulations for the relevant windows. Then analyze the updated project again. This turns the histogram into a useful feedback step: rather than treating PMF computation as the final click in a workflow, use it to identify where your next sampling effort may be needed.
GROMACS Wizard saves profiles, histograms, and plots in the project’s wham_results subfolder. Keep that output with the project so you can revisit the analysis. If you switch reaction coordinates later, the tool reuses already computed data for the same parameters.
For the full workflow and interface details, see the PMF Analysis tutorial. You may also want to review Umbrella Sampling, which generates the data used here.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.