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- PDB-9ehh: FnCas9 perfect match DNA non-productive state -

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Basic information

Entry
Database: PDB / ID: 9ehh
TitleFnCas9 perfect match DNA non-productive state
Components
  • CRISPR-associated endonuclease Cas9
  • HBB DNA NTS
  • HBB DNA TS
  • gRNA
KeywordsHydrolase/RNA/DNA / CRISPR / Cas9 / Hydrolase-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR system subtype II-B RNA-guided endonuclease Cas9/Csx12 / : / : / CRISPR-associated endonuclease Cas9 alpha-helical lobe / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9-type HNH domain / Cas9-type HNH domain profile.
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHibshman, G.N. / Taylor, D.W.
Funding support United States, 2items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural basis of a dual-function type II-B CRISPR-Cas9.
Authors: Grace N Hibshman / David W Taylor /
Abstract: Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can ...Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.
History
DepositionNov 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: gRNA
C: HBB DNA TS
D: HBB DNA NTS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)227,8306
Polymers227,7824
Non-polymers492
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: Protein CRISPR-associated endonuclease Cas9


Mass: 190756.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida (bacteria)
Gene: cas9, FTN_0757 / Production host: Escherichia coli (E. coli)
References: UniProt: A0Q5Y3, Hydrolases; Acting on ester bonds
#2: RNA chain gRNA


Mass: 27428.160 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida (bacteria)
#3: DNA chain HBB DNA TS


Mass: 6801.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: DNA chain HBB DNA NTS


Mass: 2795.847 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / 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: Ternary complex of FnCas9 with HBB gRNA and HBB DNA / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.190329 MDa / Experimental value: YES
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51749 / Symmetry type: POINT
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511656
ELECTRON MICROSCOPYf_angle_d0.9816265
ELECTRON MICROSCOPYf_dihedral_angle_d18.1842722
ELECTRON MICROSCOPYf_chiral_restr0.0561851
ELECTRON MICROSCOPYf_plane_restr0.0091661

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