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TitleAccelerated cryo-EM-guided determination of three-dimensional RNA-only structures.
Journal, issue, pagesNat Methods, Vol. 17, Issue 7, Page 699-707, Year 2020
Publish dateJul 2, 2020
AuthorsKalli Kappel / Kaiming Zhang / Zhaoming Su / Andrew M Watkins / Wipapat Kladwang / Shanshan Li / Grigore Pintilie / Ved V Topkar / Ramya Rangan / Ivan N Zheludev / Joseph D Yesselman / Wah Chiu / Rhiju Das /
PubMed AbstractThe discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve ...The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
External linksNat Methods / PubMed:32616928 / PubMed Central
MethodsEM (single particle)
Resolution4.7 - 11.0 Å
Structure data

EMDB-21831: ATP-TTR-3 with AMP
PDB-6wlj: ATP-TTR-3 with AMP models, 9.6 Angstrom resolution
Method: EM (single particle) / Resolution: 9.6 Å

EMDB-21832: Apo ATP-TTR-3
PDB-6wlk: Apo ATP-TTR-3 models, 10.0 Angstrom resolution
Method: EM (single particle) / Resolution: 10.0 Å

EMDB-21833: Apo F. nucleatum glycine riboswitch
PDB-6wll: Apo F. nucleatum glycine riboswitch models, 10.0 Angstrom resolution
Method: EM (single particle) / Resolution: 10.0 Å

EMDB-21834: F. nucleatum glycine riboswitch with glycine
PDB-6wlm: F. nucleatum glycine riboswitch with glycine models, 7.4 Angstrom resolution
Method: EM (single particle) / Resolution: 7.4 Å

EMDB-21835: hc16 ligase product
PDB-6wln: hc16 ligase product models, 10.0 Angstrom resolution
Method: EM (single particle) / Resolution: 10.0 Å

EMDB-21836: hc16 ligase
PDB-6wlo: hc16 ligase models, 11.0 Angstrom resolution
Method: EM (single particle) / Resolution: 11.0 Å

EMDB-21838: Apo SAM-IV riboswitch
PDB-6wlq: Apo SAM-IV riboswitch models, 4.7 Angstrom resolution
Method: EM (single particle) / Resolution: 4.7 Å

EMDB-21839: SAM-IV riboswitch with SAM
PDB-6wlr: SAM-IV riboswitch with SAM models, 4.8 Angstrom resolution
Method: EM (single particle) / Resolution: 4.8 Å

EMDB-21840, PDB-6wls:
Tetrahymena ribozyme models, 6.8 Angstrom resolution
Method: EM (single particle) / Resolution: 6.8 Å

EMDB-21841: Apo V. cholerae glycine riboswitch
PDB-6wlt: Apo V. cholerae glycine riboswitch models, 4.8 Angstrom resolution
Method: EM (single particle) / Resolution: 4.8 Å

EMDB-21842: V. cholerae glycine riboswitch with glycine
PDB-6wlu: V. cholerae glycine riboswitch with glycine models, 5.7 Angstrom resolution
Method: EM (single particle) / Resolution: 5.7 Å

Source
  • synthetic construct (others)
  • fusobacterium nucleatum (bacteria)
  • mycobacterium sp. (bacteria)
  • tetrahymena thermophila (eukaryote)
  • vibrio cholerae (bacteria)
KeywordsRNA / aptamer / riboswitch / ribozyme

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