Scaffold-enabled high-resolution cryo-EM structure determination of RNA.

Area of Science:

• Structural Biology
• RNA Biology
• Biochemistry

Background:

• Determining high-resolution cryo-electron microscopy (cryo-EM) structures of protein-free RNAs, especially small ones, is challenging.
• Existing methods often yield low to moderate resolution, lacking nucleotide-level detail for smaller macromolecules.

Purpose of the Study:

• To develop a novel strategy for high-resolution cryo-EM structure determination of small RNAs.
• To demonstrate the utility of this method using the thiamine pyrophosphate (TPP) riboswitch.

Main Methods:

• A strategy involving fusing small RNAs to a group II intron was employed.
• Cryo-EM was used to determine the structures of the TPP riboswitch in both ligand-bound and ligand-free states.

Main Results:

• High-resolution (2.5 Å) cryo-EM structure of the TPP riboswitch, including its ligand binding pocket, was determined.
• The structure revealed a large-scale conformational change in the aptamer domain upon ligand binding.
• The ligand-free apo state structure was also elucidated.

Conclusions:

• The developed fusion strategy enables unprecedented high-resolution cryo-EM visualization of small RNAs.
• This approach sets a new standard for riboswitch structure determination and is applicable to other previously intractable small RNAs.
• RNA-small molecule binding can induce significant conformational changes impacting gene expression.