Modular Imaging Scaffold for Single-Particle Electron MicroscopyClick to copy article linkArticle link copied!
- Nesrine AissaouiNesrine AissaouiCentre de Biochimie Structurale, CNRS UMR 5048, INSERM U1054, F-34000 Montpellier, FranceUniversité de Montpellier, F-34000 Montpellier, FranceMore by Nesrine Aissaoui
- Josephine Lai-Kee-HimJosephine Lai-Kee-HimCentre de Biochimie Structurale, CNRS UMR 5048, INSERM U1054, F-34000 Montpellier, FranceUniversité de Montpellier, F-34000 Montpellier, FranceMore by Josephine Lai-Kee-Him
- Allan MillsAllan MillsCentre de Biochimie Structurale, CNRS UMR 5048, INSERM U1054, F-34000 Montpellier, FranceUniversité de Montpellier, F-34000 Montpellier, FranceMore by Allan Mills
- Nathalie DeclerckNathalie DeclerckCentre de Biochimie Structurale, CNRS UMR 5048, INSERM U1054, F-34000 Montpellier, FranceUniversité de Montpellier, F-34000 Montpellier, FranceDepartement MICA, INRA, 78352 Jouy-en-Josas, FranceMore by Nathalie Declerck
- Zakia MorichaudZakia MorichaudUniversité de Montpellier, F-34000 Montpellier, FranceIRIM, CNRS, Université Montpellier, 1919 Route de Mende, 34293 Montpellier, FranceMore by Zakia Morichaud
- Konstantin BrodolinKonstantin BrodolinUniversité de Montpellier, F-34000 Montpellier, FranceIRIM, CNRS, Université Montpellier, 1919 Route de Mende, 34293 Montpellier, FranceMore by Konstantin Brodolin
- Sonia BaconnaisSonia BaconnaisSignalisations, Noyaux et Innovations en Cancérologie, UMR 8126, CNRS, Université Paris-Sud, Gustave Roussy, Université Paris-Saclay, 94800 Villejuif, FranceMore by Sonia Baconnais
- Eric Le CamEric Le CamSignalisations, Noyaux et Innovations en Cancérologie, UMR 8126, CNRS, Université Paris-Sud, Gustave Roussy, Université Paris-Saclay, 94800 Villejuif, FranceMore by Eric Le Cam
- Jean Baptiste CharbonnierJean Baptiste CharbonnierUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, FranceMore by Jean Baptiste Charbonnier
- Rémy SounierRémy SounierUniversité de Montpellier, F-34000 Montpellier, FranceInstitut de Génomique Fonctionnelle, CNRS UMR 5203, INSERM U1191, F-34000 Montpellier, FranceMore by Rémy Sounier
- Sébastien GranierSébastien GranierUniversité de Montpellier, F-34000 Montpellier, FranceInstitut de Génomique Fonctionnelle, CNRS UMR 5203, INSERM U1191, F-34000 Montpellier, FranceMore by Sébastien Granier
- Virginie Ropars*Virginie Ropars*E-mail: [email protected]Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, FranceMore by Virginie Ropars
- Patrick Bron*Patrick Bron*E-mail: [email protected]Centre de Biochimie Structurale, CNRS UMR 5048, INSERM U1054, F-34000 Montpellier, FranceUniversité de Montpellier, F-34000 Montpellier, FranceMore by Patrick Bron
- Gaetan Bellot*Gaetan Bellot*E-mail: [email protected]Centre de Biochimie Structurale, CNRS UMR 5048, INSERM U1054, F-34000 Montpellier, FranceUniversité de Montpellier, F-34000 Montpellier, FranceMore by Gaetan Bellot
Abstract

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.
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This article is cited by 9 publications.
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