Single particle cryo-EM map and model validation: It's not crystal clear

The application of single particle cryogenic electron microscopy (cryo-EM) to structure determination continues to have a transformative impact on our understanding on biological systems. While there has been a great deal of algorithmic development focused on improving attainable resolutions and streamlining atomic model building, there has not been commensurate development of validation metrics to ensure the accuracy of our cryo-EM maps and models. This review emphasizes the persistent issues that currently complicate single particle cryo-EM structure validation, and highlights the metrics that are gaining broad acceptance by the community. This article aims to underscore the need for further development of validation criteria and the potential role of machine learning methodologies in confidently assessing the quality of cryo-EM structures.