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- EMDB-73645: AI-generated RNA-guided nuclease R-loop formed state -

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Basic information

Entry
Database: EMDB / ID: EMD-73645
TitleAI-generated RNA-guided nuclease R-loop formed state
Map data
Sample
  • Complex: AI-designed RNA-guided nuclease R-loop formed state
    • Protein or peptide: AI-generated RNA-guided nuclease
    • RNA: RNA (132-MER)
    • DNA: DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3')
    • DNA: 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: DNA (5'-D(P*CP*CP*CP*C)-3')
KeywordsCRISPR / 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 / RNA BINDING PROTEIN
Biological speciessynthetic construct (others) / Deinococcus radiodurans (radioresistant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsSkopintsev P / Esain-Garcia I / Doudna J
Funding support United States, Switzerland, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE 2334027 United States
Swiss National Science FoundationP500PB_214418 Switzerland
CitationJournal: Science / Year: 2026
Title: Structure and evolution-guided design of minimal RNA-guided nucleases.
Authors: Petr Skopintsev / Isabel Esain-Garcia / Evan C DeTurk / Peter H Yoon / Zehan Zhou / Trevor Weiss / Maris Kamalu / Ajit Chamraj / Kenneth J Loi / Conner J Langeberg / Ron S Boger / Hunter ...Authors: Petr Skopintsev / Isabel Esain-Garcia / Evan C DeTurk / Peter H Yoon / Zehan Zhou / Trevor Weiss / Maris Kamalu / Ajit Chamraj / Kenneth J Loi / Conner J Langeberg / Ron S Boger / Hunter Nisonoff / Hannah M Karp / Lin-Xing Chen / Honglue Shi / Kamakshi Vohra / Jillian F Banfield / Jamie H D Cate / Steven E Jacobsen / Jennifer A Doudna /
Abstract: The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic ...The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
History
DepositionOct 29, 2025-
Header (metadata) releaseJul 15, 2026-
Map releaseJul 15, 2026-
UpdateJul 29, 2026-
Current statusJul 29, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73645.png

Downloads & links

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Map

FileDownload / File: emd_73645.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 280 pix.
= 237.44 Å		0.85 Å/pix.
x 280 pix.
= 237.44 Å		0.85 Å/pix.
x 280 pix.
= 237.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.848 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.0017939329 - 1.9028978
Average (Standard dev.)0.0008361847 (±0.019939467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 237.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_73645_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_73645_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : AI-designed RNA-guided nuclease R-loop formed state

EntireName: AI-designed RNA-guided nuclease R-loop formed state
Components
  • Complex: AI-designed RNA-guided nuclease R-loop formed state
    • Protein or peptide: AI-generated RNA-guided nuclease
    • RNA: RNA (132-MER)
    • DNA: DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3')
    • DNA: 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: DNA (5'-D(P*CP*CP*CP*C)-3')

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Supramolecule #1: AI-designed RNA-guided nuclease R-loop formed state

SupramoleculeName: AI-designed RNA-guided nuclease R-loop formed state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 149 KDa

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Macromolecule #1: AI-generated RNA-guided nuclease

MacromoleculeName: AI-generated RNA-guided nuclease / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 46.649828 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIHRKAFVVR LYPNARQEEL IERTLGSARF VYNHFLAERK AAYKESGKPL TYGQMSSLLT KLKQAPETAW LSEVDKFALQ NSLKNLERA FQNFFRTLKK KGEKVGFPKF RKKKTGESFR TQFTNNNIQI GKGKLKLPKL GWVKTKGQQE IKGKVLNVTV K RVHEGHYE ...String:
MIHRKAFVVR LYPNARQEEL IERTLGSARF VYNHFLAERK AAYKESGKPL TYGQMSSLLT KLKQAPETAW LSEVDKFALQ NSLKNLERA FQNFFRTLKK KGEKVGFPKF RKKKTGESFR TQFTNNNIQI GKGKLKLPKL GWVKTKGQQE IKGKVLNVTV K RVHEGHYE ASVLVEVEIP ELPEAPLLAA GVDLGIKNFA VVTDGEEFEK VENPKFLKKT EKRLARAQRR LSRRKKGSAR WE KARTRLA RIHKRIVNQR QDFLHKLTTG LVKTYQIIAV EDLNPKNMMK NHHLARAISD AAWGEFRRQL EYKAEWYGRT VAK VSRWFP SSQLCHVCGH KNPEVKDLSV RTWTCPSCGT THDRDENAAL NIRREALVAA GISDTLNAHG GYVRPASAGN GLRS ENHAT LVV

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Macromolecule #2: RNA (132-MER)

MacromoleculeName: RNA (132-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Deinococcus radiodurans (radioresistant)
Molecular weightTheoretical: 42.598207 KDa
SequenceString:
GGUGGCUGCG GGAAUCUCAG ACACCUUAAA CGCUCAUGGA GGCUAUGUCA GACCUGCUUC GGCGGGCAAU GGUCUGCGAA GUGAGAAUC ACGCGACUUU AGUCGUGUGA GGUUCAAGAG UCCCUUGGCG CCC

GENBANK: GENBANK: OV024757.1

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Macromolecule #3: DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*CP*TP*AP*TP*TP*GP*AP*T)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.416615 KDa
SequenceString:
(DC)(DT)(DA)(DT)(DT)(DG)(DA)(DT)

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Macromolecule #4: DNA (5'-D(*GP*CP*CP*AP*AP*GP*GP*GP*AP*CP*TP*CP*AP*TP*CP*AP*AP*TP*...

MacromoleculeName: DNA (5'-D(*GP*CP*CP*AP*AP*GP*GP*GP*AP*CP*TP*CP*AP*TP*CP*AP*AP*TP*AP*G)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.152007 KDa
SequenceString:
(DG)(DC)(DC)(DA)(DA)(DG)(DG)(DG)(DA)(DC) (DT)(DC)(DA)(DT)(DC)(DA)(DA)(DT)(DA)(DG)

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Macromolecule #5: DNA (5'-D(P*CP*CP*CP*C)-3')

MacromoleculeName: DNA (5'-D(P*CP*CP*CP*C)-3') / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.11177 KDa
SequenceString:
(DC)(DC)(DC)(DC)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.3.0) / Software - details: PatchCTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-initio map calculated in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.0) / Number images used: 566150
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.3.0)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

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

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