Validation tests for cryo-EM maps using an independent particle set

• Published in: Journal of structural biology. X Vol. 4; p. 100032
• Main Authors: Ortiz, Sebastian, Stanisic, Luka, Rodriguez, Boris A, Rampp, Markus, Hummer, Gerhard, Cossio, Pilar
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2020; Elsevier
• Subjects: 3D refinement; BioEM; Cryo-EM; Independent; Raw data; Reconstruction; Validation; Validation; Reconstruction; Independent; BioEM; Cryo-EM; 3D refinement; Raw data
• ISSN: 2590-1524; 2590-1524
• DOI: 10.1016/j.yjsbx.2020.100032

[Display omitted] •Use of an independent particle set to validate 3D density maps from cryo-EM.•The posterior probability of the map should increase as a function of the iteration and low-pass frequency cutoff.•The similarity between the distributions of the posterior probabilities is an indicator of the map quality.•A control particle set provides valuable information for cryo-EM map validation. Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing 3D density maps of biomolecules at near-atomic resolution. However, map validation is still an open issue. Despite several efforts from the community, it is possible to overfit 3D maps to noisy data. Here, we develop a novel methodology that uses a small independent particle set (not used during the 3D refinement) to validate the maps. The main idea is to monitor how the map probability evolves over the control set during the 3D refinement. The method is complementary to the gold-standard procedure, which generates two reconstructions at each iteration. We low-pass filter the two reconstructions for different frequency cutoffs, and we calculate the probability of each filtered map given the control set. For high-quality maps, the probability should increase as a function of the frequency cutoff and the refinement iteration. We also compute the similarity between the densities of probability distributions of the two reconstructions. As higher frequencies are included, the distributions become more dissimilar. We optimized the BioEM package to perform these calculations, and tested it over systems ranging from quality data to pure noise. Our results show that with our methodology, it possible to discriminate datasets that are constructed from noise particles. We conclude that validation against a control particle set provides a powerful tool to assess the quality of cryo-EM maps.