Scaffold-enabled high-resolution cryo-EM structure determination of RNA
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 macromo...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-01-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55699-5 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832585557179367424 |
|---|---|
| author | Daniel B. Haack Boris Rudolfs Shouhong Jin Alexandra Khitun Kevin M. Weeks Navtej Toor |
| author_facet | Daniel B. Haack Boris Rudolfs Shouhong Jin Alexandra Khitun Kevin M. Weeks Navtej Toor |
| author_sort | Daniel B. Haack |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-3a302d87812449e482d135e4e6c7b810 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-3a302d87812449e482d135e4e6c7b8102025-01-26T12:41:02ZengNature PortfolioNature Communications2041-17232025-01-0116111710.1038/s41467-024-55699-5Scaffold-enabled high-resolution cryo-EM structure determination of RNADaniel B. Haack0Boris Rudolfs1Shouhong Jin2Alexandra Khitun3Kevin M. Weeks4Navtej Toor5Department of Chemistry and Biochemistry, University of CaliforniaDepartment of Chemistry and Biochemistry, University of CaliforniaDepartment of Chemistry, University of North CarolinaDepartment of Chemistry, University of North CarolinaDepartment of Chemistry, University of North CarolinaDepartment of Chemistry and Biochemistry, University of CaliforniaAbstract 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.https://doi.org/10.1038/s41467-024-55699-5 |
| spellingShingle | Daniel B. Haack Boris Rudolfs Shouhong Jin Alexandra Khitun Kevin M. Weeks Navtej Toor Scaffold-enabled high-resolution cryo-EM structure determination of RNA Nature Communications |
| title | Scaffold-enabled high-resolution cryo-EM structure determination of RNA |
| title_full | Scaffold-enabled high-resolution cryo-EM structure determination of RNA |
| title_fullStr | Scaffold-enabled high-resolution cryo-EM structure determination of RNA |
| title_full_unstemmed | Scaffold-enabled high-resolution cryo-EM structure determination of RNA |
| title_short | Scaffold-enabled high-resolution cryo-EM structure determination of RNA |
| title_sort | scaffold enabled high resolution cryo em structure determination of rna |
| url | https://doi.org/10.1038/s41467-024-55699-5 |
| work_keys_str_mv | AT danielbhaack scaffoldenabledhighresolutioncryoemstructuredeterminationofrna AT borisrudolfs scaffoldenabledhighresolutioncryoemstructuredeterminationofrna AT shouhongjin scaffoldenabledhighresolutioncryoemstructuredeterminationofrna AT alexandrakhitun scaffoldenabledhighresolutioncryoemstructuredeterminationofrna AT kevinmweeks scaffoldenabledhighresolutioncryoemstructuredeterminationofrna AT navtejtoor scaffoldenabledhighresolutioncryoemstructuredeterminationofrna |