Separating distinct structures of multiple macromolecular assemblies from cryo-EM projections

Cryo-electron microscopy is traditionally applied to samples purified to near homogeneity as current reconstruction algorithms are unable to handle heterogeneous mixtures of structures from many macromolecular complexes. We extend on long established methods and demonstrate that relating two-dimensional projection images by their common lines in a graphical framework is sufficient for partitioning distinct protein and multiprotein complexes within the same data set. Using this approach, we first group a large set of synthetic reprojections from 35 unique macromolecular structures ranging from ∼30 – 3000 kDa into individual homogenous classes. We then apply our algorithm on cryo-EM data collected from a mixture of five protein complexes and use existing reconstruction methods to solve multiple three-dimensional structures ab initio . Incorporating methods to sort cryo-EM data from heterogeneous mixtures will alleviate the need for stringent purification and pave the way toward investigation of samples containing many unique structures.