Cross-validation is used to determine the validity of a model on unseen data by assessing if the model is overfitted to noise. It is widely used in many fields, from artificial intelligence to structural biology in X-ray crystallography and nuclear magnetic resonance. Although there are concerns of map overfitting in cryo-electron microscopy (cryo-EM), cross-validation is rarely used. The problem is that establishing a performance metric of the maps over unseen data (given by 2D-projection images) is difficult due to the low signal-to-noise ratios in the individual particles. Here, I present recent advances for cryo-EM map reconstruction. I highlight that the gold-standard procedure can fail to detect map overfitting in certain cases, showing the necessity of assessing the map quality on unbiased data. Finally, I describe the challenges and advantages of developing a robust cross-validation methodology for cryo-EM.
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