Modular Imaging Scaffold for Single-Particle Electron Microscopy
| Autor: | Joséphine Lai-Kee-Him, Nathalie Declerck, Gaëtan Bellot, Remy Sounier, Nesrine Aissaoui, Virginie Ropars, Jean-Baptiste Charbonnier, Zakia Morichaud, Patrick Bron, Sébastien Granier, Sonia Baconnais, Eric Le Cam, Konstantin Brodolin, Allan Mills |
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| Rok vydání: | 2021 |
| Předmět: |
Scaffold
Materials science Macromolecular Substances General Physics and Astronomy Nanotechnology 02 engineering and technology 010402 general chemistry 01 natural sciences chemistry.chemical_compound DNA origami General Materials Science Binding site chemistry.chemical_classification Biomolecule Cryoelectron Microscopy General Engineering DNA 021001 nanoscience & nanotechnology Single Molecule Imaging 0104 chemical sciences Microscopy Electron Membrane protein chemistry Mechanosensitive channels 0210 nano-technology Macromolecule |
| Zdroj: | ACS Nano. 15:4186-4196 |
| ISSN: | 1936-086X 1936-0851 |
| DOI: | 10.1021/acsnano.0c05113 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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