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- PDB-9yyh: AI-generated RNA-guided nuclease R-loop formed state -

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Basic information

Entry
Database: PDB / ID: 9yyh
TitleAI-generated RNA-guided nuclease R-loop formed state
Components
  • AI-generated RNA-guided nuclease
  • DNA (5'-D(*GP*CP*CP*AP*AP*GP*GP*GP*AP*CP*TP*CP*AP*TP*CP*AP*AP*TP*AP*G)-3')
  • DNA (5'-D(P*CP*CP*CP*C)-3')
  • DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3')
  • RNA (132-MER)
KeywordsRNA BINDING PROTEIN / CRISPR / nuclease / AI / Evolution Scale Modeling / Inverse Folding / ESM-IF1 / TnpB / TAM / PAM / spacer / exonuclease / DNA binding protein-DNA complex / enzyme / ribonucleoprotein / RNA / RNA-guided nuclease / Cas12 / ISDra2 / ESM
Function / homology: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
Deinococcus radiodurans (radioresistant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsSkopintsev, P. / Esain-Garcia, I. / Doudna, J.
Funding support United States, Switzerland, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE 2334027 United States
Swiss National Science FoundationP500PB_214418 Switzerland
CitationJournal: To Be Published
Title: Structure and evolution-guided design of minimal RNA-guided nucleases
Authors: Skopintsev, P. / Esain-Garcia, I. / Doudna, J.
History
DepositionOct 29, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 15, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: AI-generated RNA-guided nuclease
B: RNA (132-MER)
C: DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3')
D: DNA (5'-D(*GP*CP*CP*AP*AP*GP*GP*GP*AP*CP*TP*CP*AP*TP*CP*AP*AP*TP*AP*G)-3')
E: DNA (5'-D(P*CP*CP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)98,9285
Polymers98,9285
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein AI-generated RNA-guided nuclease


Mass: 46649.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / References: Hydrolases; Acting on ester bonds
#2: RNA chain RNA (132-MER)


Mass: 42598.207 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus radiodurans (radioresistant)
Production host: Escherichia coli (E. coli) / References: GenBank: 2155018471
#3: DNA chain DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3')


Mass: 2416.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(*GP*CP*CP*AP*AP*GP*GP*GP*AP*CP*TP*CP*AP*TP*CP*AP*AP*TP*AP*G)-3')


Mass: 6152.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (5'-D(P*CP*CP*CP*C)-3')


Mass: 1111.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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: AI-designed RNA-guided nuclease R-loop formed state / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.149 MDa / Experimental value: NO
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.3.0particle selection
2SerialEM3.8.7image acquisition
4cryoSPARC4.3.0CTF correctionPatchCTF
9PHENIX1.21.1-5419model refinement
10cryoSPARC4.3.0initial Euler assignment
11cryoSPARC4.3.0final Euler assignment
12cryoSPARC4.3.0classification
13cryoSPARC4.3.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 566150 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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