Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures

Details

• Alternative Title:
  Nat Methods
• Personal Author:
  Kappel, Kalli ; Zhang, Kaiming ; Su, Zhaoming ; Watkins, Andrew M. ; Kladwang, Wipapat ; Li, Shanshan ; Pintilie, Grigore ; Topkar, Ved V. ; Rangan, Ramya ; Zheludev, Ivan N. ; Yesselman, Joseph D. ; Chiu, Wah ; Das, Rhiju
• Description:
  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.
• Subjects:
  Article
• Source:
  Nat Methods. 17(7):699-707
• Pubmed ID:
  32616928
• Pubmed Central ID:
  PMC7386730
• Document Type:
  Journal Article
• Funding:
  P41GM103832/U.S. Department of Health & Human Services ; National Institutes of Health (NIH)/ ; U54 GM103297/GM/NIGMS NIH HHS/United States ; GRFP/National Science Foundation (NSF)/ ; U54GM103297/U.S. Department of Health & Human Services ; National Institutes of Health (NIH)/ ; R01GM079429/U.S. Department of Health & Human Services ; National Institutes of Health (NIH)/ ; S10 OD021600/CD/ODCDC CDC HHS/United States ; P01AI120943/U.S. Department of Health & Human Services ; National Institutes of Health (NIH)/ ; P41 GM103832/GM/NIGMS NIH HHS/United States ; R01 GM079429/GM/NIGMS NIH HHS/United States ; R35 GM112579/U.S. Department of Health & Human Services ; National Institutes of Health (NIH)/ ; S10 OD021600/OD/NIH HHS/United States ; R21 AI145647/AI/NIAID NIH HHS/United States ; R35 GM122579/GM/NIGMS NIH HHS/United States
• Volume:
  17
• Issue:
  7
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:3e6d95c204ef6cacacb9685fb23213209391e3885598fd5fe87005bffdd25da6
• Download URL:
  https://stacks.cdc.gov/view/cdc/91178/cdc_91178_DS1.pdf
• File Type:
  [PDF - 5.06 MB ]