In stage 1 (yellow), the starting structure is subjected to initial equilibration in explicit solvent. Equilibration at = 300 K for 50 ns is needed to drive the starting structure further away from the target state, posing an additional refinement challenge. In real applications, a short equilibration run at = 100 K for 5–10 ns should be sufficient. Stages 2 and 3 (gray and green) include the refinement against one of the half-maps (training map) followed by cross-validation by means of Fourier Shell Correlation (FSC) against the other half-map (validation map). In case no overfitting of the training map is observed, the final structure from the half-map refinement is passed to stage 4 (cyan), where it is refined against the full map to account for high-resolution features not present in both half-maps. Finally, in stage 5 (purple), the average structure from the last 5 ns of the full map refinement is subjected to geometry and goodness-of-fit assessment. Note that the procedure is automated such that stages 1–4 can be run without human intervention. Pausing the refinement to inspect intermediate results or introduce changes to the protocol should be possible at any stage. See Materials and methods for more details.