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- PDB-9sjl: Type I-F_HNH variant Cascade bound to dsDNA, HNH domain in inward... -

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Basic information

Entry
Database: PDB / ID: 9sjl
TitleType I-F_HNH variant Cascade bound to dsDNA, HNH domain in inwards position
Components
  • Cas5f
  • Cas6f
  • Cas7f
  • Cas8f
  • Non-target strand
  • Target strand
  • crRNA
KeywordsRNA BINDING PROTEIN / CRISPR-Cas Type I-F HNH nuclease
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / nucleic acid binding / zinc ion binding
Similarity search - Function
CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / HNH endonuclease / HNH endonuclease / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / HNH nucleases ...CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / HNH endonuclease / HNH endonuclease / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / HNH nucleases / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / HNH nuclease
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Cas5f / Cas6f / Cas7f / Cas8f fusion with HNH
Similarity search - Component
Biological speciesSelenomonas sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.93 Å
AuthorsFuglsang, A. / Montoya, G.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.
Authors: Anders Fuglsang / Sweta Suman Rout / Eliska Bartl Koutna / Nicholas Sofos / Alejandro Redondo Gallego / Guillermo Montoya /
Abstract: The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous ...The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.
History
DepositionAug 31, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 18, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 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

Downloads & links

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Assembly

Deposited unit
A: Cas8f
B: Cas5f
I: Cas6f
L: Non-target strand
D: Cas7f
E: Cas7f
F: Cas7f
C: Cas7f
G: Cas7f
H: Cas7f
K: Target strand
J: crRNA


Theoretical massNumber of molelcules
Total (without water)368,74812
Polymers368,74812
Non-polymers00
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

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Protein , 4 types, 9 molecules ABIDEFCGH

#1: Protein Cas8f / CRISPR-associated protein Cas8f / HNH endonuclease


Mass: 39339.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0AAX7FM29
#2: Protein Cas5f / CRISPR-associated protein Cas5f


Mass: 28703.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0AAX7FM22
#3: Protein Cas6f / CRISPR-associated protein Cas6f


Mass: 20735.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0AAX7FM27
#5: Protein
Cas7f / CRISPR-associated protein Cas7f


Mass: 38700.172 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0AAX7FM28

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DNA chain , 2 types, 2 molecules LK

#4: DNA chain Non-target strand


Mass: 14233.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#6: DNA chain Target strand


Mass: 14094.042 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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RNA chain , 1 types, 1 molecules J

#7: RNA chain crRNA


Mass: 19440.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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Details

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: Type I-F_HNH effector complex bound to dsDNA / Type: COMPLEX / Entity ID: #3, #1 / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Selenomonas sp. (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2883 / Symmetry type: POINT
RefinementCross valid method: NONE

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