Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly

Area of Science:

• Structural Biology
• Molecular Biology
• Biochemistry

Background:

• High-resolution structural studies are crucial for understanding RNA folding and function.
• Current methods for RNA structure determination face challenges with flexibility and size.

Purpose of the Study:

• To present a novel nanoarchitectural engineering strategy, ROCK, for efficient structural determination of RNA-only structures using single-particle cryo-EM.
• To enable de novo model building of complete RNA structures, including previously uncharacterized domains.

Main Methods:

• ROCK involves installing kissing-loop sequences onto functionally nonessential RNA stems.
• This promotes homomeric self-assembly into closed rings, increasing molecular weight and reducing flexibility.
• Single-particle cryo-electron microscopy (cryo-EM) is used for structural determination.

Main Results:

• ROCK enabled high-resolution cryo-EM reconstruction of the Tetrahymena group I intron (2.98 Å overall resolution).
• This allowed de novo model building of the complete intron RNA, including peripheral domains.
• ROCK was successfully applied to the Azoarcus group I intron and FMN riboswitch, revealing conformational changes and ligand binding.

Conclusions:

• ROCK is a powerful strategy for facilitating RNA structural studies using cryo-EM.
• This method significantly improves the efficiency and resolution of RNA structure determination.
• ROCK holds promise for advancing our understanding of diverse RNA structures and functions.