A molecular dynamics run can be ready to start and still leave you wondering whether the system is actually prepared for the next step. In NVT equilibration, the key question is not whether the temperature trace is perfectly flat—it is whether the system’s temperature is fluctuating around the target and its running behavior looks stable enough to continue.
This walkthrough focuses on a common workflow problem: how to set up NVT, read the temperature plot, and decide what to do if the target has not stabilized yet. NVT keeps the number of particles, volume, and temperature constant. It follows energy minimization and comes before NPT equilibration, which is used to stabilize density.
Start from a sound input
Begin with a minimized system. Before launching NVT, check that the minimized structure looks reasonable and that the minimization output has no unresolved problems. GROMACS Wizard can take either a GRO file from minimization (or a previous equilibration run) or a suitable batch project. If you are continuing from the previous step, the auto-fill control can populate the input path from the last successful run; otherwise, select the GRO file yourself.
Keep the first run straightforward
The NVT tab includes commonly adjusted settings such as the integration time step and number of steps, while additional molecular dynamics parameters are available through All…. The default values are intended for typical NVT runs. For a first pass, the tutorial recommends restoring the parameters to their defaults rather than changing several settings at once. That makes it easier to interpret the result and reduces the chance that a parameter mismatch is the source of a problem.
Temperature coupling deserves particular attention. The documentation gives velocity rescaling with a stochastic term (v-rescale) and a time constant of about 1 ps as a typical choice for many projects. Crucially, the temperature used for velocity generation should correspond to the coupling temperature. If you couple multiple groups—for example, Protein and non-Protein—make sure the number of time constants and reference temperatures matches the number of groups.
Give the system enough time, then judge the trend
The time needed depends on the size and contents of the system. A typical starting range is 50–100 ps, but treat that as a guide, not a guarantee. The useful signal is the running average: it should approach a plateau around the desired temperature. Individual fluctuations are normal, so a jagged trace alone does not mean the run has failed.
After the job finishes, inspect the temperature plot in the NVT tab. In the tutorial example, the temperature stabilizes around the default target of 300 K. Compare your plot with the target you actually configured, rather than assuming every project should be at 300 K.
If it is not stable yet, continue from the latest result
If the target has not been reached or the running average has not settled, the next step is often simply more NVT time. Run the equilibration again using the output from the previous NVT run as the input. This continues the preparation from the current state instead of returning to the earlier minimized structure.
GROMACS Wizard can generate inputs for another environment, run locally, or launch a cloud job. For a local run, progress is available in the Output window and the Local jobs list. Once the job is complete, choose whether to import the full trajectory, the last frame, or a frame range. If you cancel the import, the results are not deleted; the plot can still be generated.
Know when to move on
Continue to NPT only when the temperature is stable enough for your workflow. Small fluctuations around the target are expected; the practical checkpoint is a stable-looking running behavior, not a perfectly constant line. NPT is the subsequent step for bringing the system toward the desired density.
For the complete interface walkthrough and related parameter details, read the NVT Equilibration documentation.
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