Modular Imaging Scaffold for Single-Particle Electron Microscopy

Nesrine Aissaoui, Josephine Lai-Kee-Him, Allan Mills, Nathalie Declerck, Zakia Morichaud, Konstantin Brodolin, Sonia Baconnais, Eric Le Cam, Jean Baptiste Charbonnier, Rémy Sounier, Sébastien Granier, Virginie Ropars, Patrick Bron, Gaetan Bellot

Résultats de recherche: Contribution à un journalArticleRevue par des pairs

9 Citations (Scopus)

Résumé

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.

langue originaleAnglais
Pages (de - à)4186-4196
Nombre de pages11
journalACS Nano
Volume15
Numéro de publication3
Les DOIs
étatPublié - 23 mars 2021
Modification externeOui

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