Modular Imaging Scaffold for Single-Particle Electron Microscopy.
ACS Nano
; 15(3): 4186-4196, 2021 03 23.
Article
em En
| MEDLINE
| ID: mdl-33586425
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Imagem Individual de Molécula
Idioma:
En
Revista:
ACS Nano
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
França