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- PDB-9ehx: FnCas9 16 mismatch DNA product state -

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ID or keywords:

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Basic information

Entry
Database: PDB / ID: 9ehx
TitleFnCas9 16 mismatch DNA product state
Components
  • CRISPR-associated endonuclease Cas9
  • HBB DNA NTS
  • HBB DNA TS 1
  • HBB DNA TS 2
  • gRNA
KeywordsHydrolase/RNA/DNA / CRISPR / Cas9 / IMMUNE SYSTEM / 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: Hibshman, G.N. / Taylor, D.W.
History
DepositionNov 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: HBB DNA TS 1
D: HBB DNA NTS
A: CRISPR-associated endonuclease Cas9
B: gRNA
c: HBB DNA TS 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)232,7797
Polymers232,7315
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

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DNA chain , 3 types, 3 molecules CDc

#1: DNA chain HBB DNA TS 1


Mass: 3647.393 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: DNA chain HBB DNA NTS


Mass: 2795.847 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain HBB DNA TS 2


Mass: 5491.566 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Protein / RNA chain / Non-polymers , 3 types, 4 molecules AB

#3: 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
#4: RNA chain gRNA


Mass: 30039.703 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida (bacteria)
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

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 16 MM HBB DNA
Type: COMPLEX / Entity ID: #1-#5 / 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79858 / 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_d0.00514561
ELECTRON MICROSCOPYf_angle_d1.01920229
ELECTRON MICROSCOPYf_dihedral_angle_d17.523248
ELECTRON MICROSCOPYf_chiral_restr0.0562279
ELECTRON MICROSCOPYf_plane_restr0.0082131

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