Deciphering a hexameric protein complex with Angstrom optical resolution

Abstract

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases, where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and the hexamer geometry of Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic, environmental and dynamic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology. Significance statementFluorescence super-resolution microscopy has witnessed many clever innovations in the last two decades. Here, we advance the frontiers of this field of research by combining partial labeling and 2D image classification schemes with polarization-encoded single-molecule localization at liquid helium temperature to reach Angstrom resolution in three dimensions. We demonstrate the performance of the method by applying it to trimer and hexamer protein complexes. Our approach holds great promise for examining membrane protein structural assemblies and conformations in challenging native environments. The methodology closes the gap between electron and optical microscopy and offers an ideal ground for correlating the two modalities at the single-particle level. Indeed, correlative light and electron microscopy is an emerging technique that will provide new insight into cell biology.