Scaffold engineering enables high-resolution cryo-EM analysis of thiamine pyrophosphate riboswitch architecture and dynamics.

Cryogenic electron microscopy (cryo-EM) is well suited for studying dynamic macromolecules, but has historically been incompatible with small protein-free RNAs like thiamine pyrophosphate (TPP) riboswitches. This chapter describes a method utilizing an RNA scaffold derived from the Oceanobacillus iheyensis group II intron that overcomes these barriers, allowing for high-resolution reconstructions of an embedded target RNA. This approach produced the first reconstruction of the native open conformation of the apo E. coli TPP riboswitch aptamer, as well as a high-resolution reconstruction of the bound state. The adaptability of the scaffold design allows for any target RNA sequence to easily be embedded for structure determination by cryo-EM. This chapter outlines protocols for construct design parameters, sample preparation, and the unique data processing steps required to use this scaffold to characterize the structures of TPP riboswitch aptamer variants, folding intermediates, and bound to alternate small-molecule aptamer binders.