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- PDB-9mme: ROOLfirm-octamer-wild type -

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Basic information

Entry
Database: PDB / ID: 9mme
TitleROOLfirm-octamer-wild type
ComponentsRNA (523-MER)
KeywordsRNA / natural RNA nanocage / cryo-EM / drug delivery
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesFirmicutes bacterium CAG:227 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsLing, X.B. / Ma, J.B. / Fang, W.W. / Korostelev, A.A.
Funding support China, United States, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)NSFC 31971130 China
National Natural Science Foundation of China (NSFC)32471347 China
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM127094 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM150953 United States
CitationJournal: Nature / Year: 2025
Title: Cryo-EM structure of a natural RNA nanocage.
Authors: Xiaobin Ling / Dmitrij Golovenko / Jianhua Gan / Jinbiao Ma / Andrei A Korostelev / Wenwen Fang /
Abstract: Long (>200 nucleotides) non-coding RNAs (lncRNAs) play important roles in diverse aspects of life. Over 20 classes of lncRNAs have been identified in bacteria and bacteriophages through comparative ...Long (>200 nucleotides) non-coding RNAs (lncRNAs) play important roles in diverse aspects of life. Over 20 classes of lncRNAs have been identified in bacteria and bacteriophages through comparative genomics analyses, but their biological functions remain largely unexplored. Owing to the large sizes, the structural determinants of most lncRNAs also remain uncharacterized. Here, we report the structures of two natural RNA nanocages formed by the ROOL (rumen-originating, ornate, large) lncRNA found in bacterial and phage genomes. The cryo-electron microscopy (cryo-EM) structures at 2.9-Å resolution reveal that ROOL RNAs form an octameric nanocage with a diameter of 28 nm and an axial length of 20 nm, in which the hollow inside features poorly ordered regions. The octamer is stabilized by numerous tertiary and quaternary interactions, including triple-strand A-minors, for which we propose the term 'A-minor staples'. The structure of an isolated ROOL monomer at 3.2-Å resolution indicates that nanocage assembly involves a strand-swapping mechanism resulting in quaternary kissing loops. Finally, we show that ROOL RNA fused to an RNA aptamer, transfer RNA or microRNA retains its structure, forming a nanocage with radially displayed cargoes. Our findings, therefore, may enable engineering of novel RNA nanocages as delivery vehicles for research and therapeutic applications.
History
DepositionDec 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
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Revision 1.0Jun 4, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.1Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Sep 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
U: RNA (523-MER)
Y: RNA (523-MER)
c: RNA (523-MER)
g: RNA (523-MER)
k: RNA (523-MER)
o: RNA (523-MER)
s: RNA (523-MER)
w: RNA (523-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,504,918105
Polymers1,501,7328
Non-polymers3,18697
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain
RNA (523-MER)


Mass: 187716.484 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Firmicutes bacterium CAG:227 (bacteria)
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 41 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical...
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 56 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: lncRNA-ROOL1-octamer / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Firmicutes bacterium CAG:227 (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 51.23 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 420040 / Symmetry type: POINT
RefinementHighest resolution: 2.93 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007100087
ELECTRON MICROSCOPYf_angle_d0.869156030
ELECTRON MICROSCOPYf_dihedral_angle_d13.8449944
ELECTRON MICROSCOPYf_chiral_restr0.05120840
ELECTRON MICROSCOPYf_plane_restr0.0074168

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