Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution

Abstract

Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

Fig. 1

Single-particle cryo-EM analysis of the apo SAM-IV riboswitch collected on Titan Krios. a Representative motion-corrected cryo-EM micrograph. Scale bar represents 100 Å. b Reference-free 2D class averages computed in Relion. c Final 3D reconstruction in two different views.

Fig. 2

The secondary and tertiary structures of the apo SAM-IV riboswitch. a The secondary structure with the domains colored differently. The black arrow indicates the direction of the backbone. b-c The model and map colored by the same scheme as in a. P paired, PK pseudoknot.

Fig. 3

Determination of the ligand location in the ligand-bound SAM-IV cryo-EM map. a Ligand-bound SAM-IV cryo-EM map. b Map computed from the SAM-IV RNA-only model from a. c Difference map between a and b shown at two different thresholds. The insets show the prominent density in the difference map into which are fitted the ligand model derived from SAM-I crystal structure (PDB code: 3GX5). d Segmented SAM density from a is highlighted in a wire frame representation, fitted with the ligand model derived from SAM-I crystal structure (PDB code: 3GX5). Q-scores of the ligand components between the map and model are calculated.