Umbrella sampling turns a molecular process into a set of simulations centered on different positions along a reaction coordinate. The next challenge is bringing those windows together: how do you turn their data into one Potential of Mean Force (PMF) profile, and how can you tell whether the result is supported across the coordinate range?
In GROMACS Wizard, the WHAM Analysis tab uses the Weighted Histogram Analysis Method (WHAM) to calculate the PMF from umbrella-sampling data. The result is useful to inspect as a pair: a PMF profile and a histogram showing how the reaction-coordinate space is covered. Looking at both helps you avoid treating a curve as the whole story.
Start with a consistent set of windows
Before running the analysis, check that the input folder contains numbered subfolders belonging to the same system and the same reaction-coordinate setup. WHAM combines data across windows, so mixing projects or coordinate definitions is not a good starting point.
Open GROMACS Wizard and switch to WHAM Analysis. Select the project path, or use auto-fill if your previous project was the umbrella-sampling simulation. Confirm the folder organization before proceeding. GROMACS Wizard loads reaction-coordinate information, the time range, and temperature from the project automatically.
Run the analysis with deliberate settings
Choose the reaction coordinate you want to analyze. If your analysis calls for them, adjust the optional custom bounds, time range, or energy units. Then click Compute. For large trajectories, allow several seconds or minutes for the calculation to finish.
The output includes two plots: the PMF profile and a histogram of coverage along the coordinate. The profile is the result many people focus on first, but the histogram answers a practical companion question: where do the input windows provide coverage, and where might more sampling be needed?
Read the histogram alongside the profile
Use the histogram to inspect whether the reaction-coordinate space is covered across the region you care about. A region with weaker coverage is a prompt to review the corresponding windows rather than simply relying on the appearance of the PMF curve. If sampling appears insufficient in some regions, the documented next step is to return to umbrella sampling and add or extend simulations for those windows, then analyze the resulting data again.
This paired review is a useful habit: the PMF plot shows the calculated profile, while the histogram helps you assess how the coordinate space was sampled. Neither plot should be read in isolation when deciding whether the analysis is ready to interpret.
Keep results organized for later checks
GROMACS Wizard saves profiles, histograms, and plots in the project’s wham_results subfolder. If you later switch reaction coordinates, it reuses data already computed for the same parameters. Keeping the project and its output together makes it easier to revisit the analysis and compare the plots with the input windows.
In short: verify that the windows belong together, select the intended coordinate, compute the PMF, and inspect coverage before drawing conclusions. For the full workflow and interface details, see the GROMACS Wizard PMF Analysis documentation.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.