Gold-standard local validation enhances interpretability of cryo-EM maps

Accurate interpretation of cryo-electron microscopy (cryo-EM) maps requires robust local measures of quality and resolution. Current approaches are often limited by single-map estimates, user-defined solvent masks that risk bias and overfitting, and an exclusive reliance on signal amplitudes while neglecting phase information. Here, we present a Gold-standard framework requiring minimal user intervention for local validation and enhancement of cryo-EM reconstructions that addresses these limitations by jointly analyzing amplitudes and phases between independent half-maps or between maps and atomic models. All methods are computed voxel-wise in real space from independent half-maps, ensuring unbiased local evaluation. The toolbox includes unbiased local sharpening (LocSpiral2), per-voxel figure-of-merit, signal-to-noise estimation and general quality assessment (LocFOM, LocSNR and LocQ), directional local anisotropy evaluation (LocAnisotropy) and Gold-standard local resolution mapping (LocResMap). Together, these approaches provide a comprehensive and unified strategy for evaluating cryo-EM maps at the voxel level, improving interpretability and offering more reliable guidance for atomic modelling. Applications to challenging experimental datasets demonstrate enhanced map sharpening, robust local validation, and improved structural insights.