Deciphering a hexameric protein complex with Angstrom optical resolution

Mazal H, Wieser FF, Sandoghdar V (2022)

Publication Type: Journal article

Publication year: 2022

Journal

eLife eLife Sciences Publications

Book Volume: 11

DOI: 10.7554/eLife.76308

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 six different sites of the hexamer protein 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 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.

Authors with CRIS profile

Vahid Sandoghdar Lehrstuhl für Experimentalphysik

Involved external institutions

Max-Planck-Institut für die Physik des Lichts (MPL) / Max Planck Institute for the Science of Light DE Germany (DE)

How to cite

APA:

Mazal, H., Wieser, F.-F., & Sandoghdar, V. (2022). Deciphering a hexameric protein complex with Angstrom optical resolution. eLife, 11. https://dx.doi.org/10.7554/eLife.76308

MLA:

Mazal, Hisham, Franz-Ferdinand Wieser, and Vahid Sandoghdar. "Deciphering a hexameric protein complex with Angstrom optical resolution." eLife 11 (2022).

BibTeX: Download