Cryo-EM structure of a 40\xa0kDa SAM-IV riboswitch RNA at 3.7\u2009\xc5 resolution

Kaiming Zhang,Shanshan Li,Wah Chiu,Rhiju Das,Zhaoming Su,Michael F Schmid,Kalli Kappel,Grigore Pintilie,Tung-Chung Mou

doi:10.1038/s41467-019-13494-7

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.

Highlights

Specimens below 50 kDa have generally been considered too small to be analyzed by singleparticle cryo-electron microscopy
S-adenosylmethionine (SAM), a cofactor used in many methylation reactions, is recognized by seven classes of riboswitches, including SAM-I, SAM-II, SAM-III, SAM-IV, SAM-I/IV, SAM-V, and SAM-VI6
This study reveals the structural basis of ligand recognition by the SAM-IV riboswitch and demonstrates the feasibility of cryo-electron microscopy (cryo-EM) for structure determination below the current size limit and detection of ligandbinding sites

Summary

Introduction

Specimens below 50 kDa have generally been considered too small to be analyzed by singleparticle cryo-electron microscopy (cryo-EM). 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. Metabolite-binding riboswitches are non-coding RNAs that bind to metabolites with high specificity and regulate downstream gene expression in response to changes in metabolite concentrations[2]. These molecules are of growing interest as potential targets for new classes of antibiotics[3,4,5]. We use cryo-EM to determine maps of both apo and ligand-bound SAM-IV riboswitches (119-nt, ~40 kDa) from Mycobacterium sp. This study reveals the structural basis of ligand recognition by the SAM-IV riboswitch and demonstrates the feasibility of cryo-EM for structure determination below the current size limit and detection of ligandbinding sites

Methods

Results

Conclusion