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- PDB-9jwk: Cryo-EM structure of FrCas9 in complex with sgRNA and 26-nt TS an... -

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Basic information

Entry
Database: PDB / ID: 9jwk
TitleCryo-EM structure of FrCas9 in complex with sgRNA and 26-nt TS and 4-nt NTS substrates
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (26-mer)
  • sgRNA
KeywordsHYDROLASE/RNA/DNA / FrCas9 / high fidelity / off-target / nuclease protein / complex / HYDROLASE / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesFaecalibaculum rodentium (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsChen, S.D. / Yang, M. / Liu, S.Q.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: To Be Published
Title: Structural basis of Faecalibaculum rodentium Cas9 with high genome editing efficiency and fidelity provide insights into protein engineering.
Authors: Chen, S.D. / Yang, M. / Liu, S.Q.
History
DepositionOct 10, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data 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: CRISPR-associated endonuclease Cas9
B: sgRNA
D: DNA (26-mer)


Theoretical massNumber of molelcules
Total (without water)207,6763
Polymers207,6763
Non-polymers00
Water181
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 CRISPR-associated endonuclease Cas9 / FrCas9


Mass: 159661.672 Da / Num. of mol.: 1 / Mutation: H877A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Faecalibaculum rodentium (bacteria) / Gene: cas9, BO223_00085 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A1Q9YNK3, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA / RNA (125-mer)


Mass: 40071.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Faecalibaculum rodentium (bacteria)
#3: DNA chain DNA (26-mer)


Mass: 7943.136 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
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 FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Faecalibaculum rodentium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
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: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219843 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312931
ELECTRON MICROSCOPYf_angle_d0.58518084
ELECTRON MICROSCOPYf_dihedral_angle_d15.0512899
ELECTRON MICROSCOPYf_chiral_restr0.0372091
ELECTRON MICROSCOPYf_plane_restr0.0051803

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