Liu, DiThelot, FrancoisLiao, MaofuYin, Peng2023-06-292022-05-02Liu, Di, François A Thélot, Joseph A Piccirilli, Maofu Liao, and Peng Yin. 2022. “Sub-3-Å Cryo-EM Structure of RNA Enabled by Engineered Homomeric Self-Assembly.” Nature Methods 19 (5): 576–85.https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37376306Though many functional RNAs fold into intricate and precise 3D architectures, it is difficult to acquire their high-resolution structures. Herein, we present a nanoarchitectural strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy, termed RNA oligomerization-enabled cryo-EM via installing kissing-loops (ROCK), involves RNA construct engineering to install kissing-loop sequences onto functionally nonessential stems for the homomeric self-assembly into closed nanoarchitectures with multiplied molecular weights and mitigated structural flexibility. ROCK enables the cryo-EM reconstruction of the Tetrahymena group I intron at 2.98 Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA including the previously unknown peripheral domains. ROCK is also applied to two smaller RNAs to produce modest-resolution maps, revealing the conformational change of the Azoarcus group I intron and the bound ligand in the FMN riboswitch. Our work unleashes the largely unexplored potential of cryo-EM in RNA structural studies.en-USJournal Article2023-06-2910.1101/2021.08.11.455951