Abstract:Cryo-electron microscopy (cryo-EM) is a powerful technique for determining high-resolution 3D biomolecular structures from imaging data. As this technique can capture dynamic biomolecular complexes, 3D reconstruction methods are increasingly being developed to resolve this intrinsic structural heterogeneity. However, the absence of standardized benchmarks with ground truth structures and validation metrics limits the advancement of the field. Here, we propose CryoBench, a suite of datasets, metrics, and performance benchmarks for heterogeneous reconstruction in cryo-EM. We propose five datasets representing different sources of heterogeneity and degrees of difficulty. These include conformational heterogeneity generated from simple motions and random configurations of antibody complexes and from tens of thousands of structures sampled from a molecular dynamics simulation. We also design datasets containing compositional heterogeneity from mixtures of ribosome assembly states and 100 common complexes present in cells. We then perform a comprehensive analysis of state-of-the-art heterogeneous reconstruction tools including neural and non-neural methods and their sensitivity to noise, and propose new metrics for quantitative comparison of methods. We hope that this benchmark will be a foundational resource for analyzing existing methods and new algorithmic development in both the cryo-EM and machine learning communities.