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- PDB-9jwn: Cryo-EM structure of FrCas9 in complex with sgRNA and 43-bp dsDNA... -

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Basic information

Entry
Database: PDB / ID: 9jwn
TitleCryo-EM structure of FrCas9 in complex with sgRNA and 43-bp dsDNA substrate
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (13-mer)
  • DNA (43-mer)
  • RNA (125-mer)
KeywordsDNA/RNA/HYDROLASE / FrCas9 / high fidelity / off-target / nuclease protein / complex / HYDROLASE / DNA-RNA-HYDROLASE 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 speciesHomo sapiens (human)
Faecalibaculum rodentium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsChen, S.D. / Yang, M. / Liu, S.Q.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Genom / Year: 2025
Title: Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.
Authors: Mei Yang / Siqi Liu / Guanqiao Chen / Xi Liu / Dapeng Sun / Jingjing Zhang / Yumei Wang / Shoudeng Chen / Rui Tian / Zheng Hu /
Abstract: The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic ...The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.
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
Revision 1.1Oct 29, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: DNA (13-mer)
B: RNA (125-mer)
C: DNA (43-mer)
A: CRISPR-associated endonuclease Cas9


Theoretical massNumber of molelcules
Total (without water)216,9994
Polymers216,9994
Non-polymers00
Water724
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain DNA (13-mer)


Mass: 3996.619 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#2: RNA chain 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 (43-mer)


Mass: 13269.536 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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 a 43-bp dsDNA
Type: COMPLEX / Entity ID: #1-#4 / 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: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 684922 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312484
ELECTRON MICROSCOPYf_angle_d0.51917545
ELECTRON MICROSCOPYf_dihedral_angle_d18.6532935
ELECTRON MICROSCOPYf_chiral_restr0.0342033
ELECTRON MICROSCOPYf_plane_restr0.0041675

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