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

Author

Listed:
  • Daniel B. Haack

    (University of California)

  • Boris Rudolfs

    (University of California)

  • Shouhong Jin

    (University of North Carolina)

  • Alexandra Khitun

    (University of North Carolina)

  • Kevin M. Weeks

    (University of North Carolina)

  • Navtej Toor

    (University of California)

Abstract

Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-nt raiA bacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure of raiA at 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.

Suggested Citation

  • Daniel B. Haack & Boris Rudolfs & Shouhong Jin & Alexandra Khitun & Kevin M. Weeks & Navtej Toor, 2025. "Scaffold-enabled high-resolution cryo-EM structure determination of RNA," Nature Communications, Nature, vol. 16(1), pages 1-17, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-024-55699-5
    DOI: 10.1038/s41467-024-55699-5
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    References listed on IDEAS

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    1. Zhaoming Su & Kaiming Zhang & Kalli Kappel & Shanshan Li & Michael Z. Palo & Grigore D. Pintilie & Ramya Rangan & Bingnan Luo & Yuquan Wei & Rhiju Das & Wah Chiu, 2021. "Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution," Nature, Nature, vol. 596(7873), pages 603-607, August.
    2. Alexander Serganov & Anna Polonskaia & Anh Tuân Phan & Ronald R. Breaker & Dinshaw J. Patel, 2006. "Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch," Nature, Nature, vol. 441(7097), pages 1167-1171, June.
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    4. Aaron R. Robart & Russell T. Chan & Jessica K. Peters & Kanagalaghatta R. Rajashankar & Navtej Toor, 2014. "Crystal structure of a eukaryotic group II intron lariat," Nature, Nature, vol. 514(7521), pages 193-197, October.
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