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- PDB-9vjb: Type II-A CRISPR integrase pre-integration complex -

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Basic information

Entry
Database: PDB / ID: 9vjb
TitleType II-A CRISPR integrase pre-integration complex
Components
  • (CRISPR-associated ...) x 2
  • (DNA (51-MER)) x 2
  • Type II-A CRISPR-associated protein Csn2
KeywordsDNA BINDING PROTEIN/DNA / CRISPR / Cas1 / Cas2 / Csn2 / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / DNA binding / metal ion binding
Similarity search - Function
CRISPR-associated protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1
Similarity search - Domain/homology
DNA / DNA (> 10) / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1 / Type II-A CRISPR-associated protein Csn2
Similarity search - Component
Biological speciesEnterococcus faecalis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsLi, Z.X. / Li, Y.T. / Lu, M.L. / Xiao, Y.B.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970547 China
Ministry of Science and Technology (MoST, China)2018YFA0902000 China
National Natural Science Foundation of China (NSFC)82304614 China
CitationJournal: Mol Cell / Year: 2026
Title: Structural basis for Cas9-directed spacer acquisition in type II-A CRISPR-Cas systems.
Authors: Zhaoxing Li / Yutao Li / Jianping Kong / Qianqian Wu / Pingping Huang / Yu Zhang / Wanqian Wu / Meirong Chen / Yongfeng Liu / HanFeng Lin / Liqiu Hou / Gongyu Liu / Ting Zeng / Yutong He / ...Authors: Zhaoxing Li / Yutao Li / Jianping Kong / Qianqian Wu / Pingping Huang / Yu Zhang / Wanqian Wu / Meirong Chen / Yongfeng Liu / HanFeng Lin / Liqiu Hou / Gongyu Liu / Ting Zeng / Yutong He / Chunyi Hu / Zhenhuang Yang / Meiling Lu / Min Luo / Yibei Xiao /
Abstract: CRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate ...CRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-electron microscopy (cryo-EM) structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo state (Cas9₂-Csn2₈-Cas1₈-Cas2₄) is a resting complex, while DNA binding forms a prespacer-catching complex threading DNA through Csn2's channel, enabling Cas9 to interrogate the PAM sequence while sliding along the DNA. Cas9 and Csn2 jointly define a 30-bp DNA segment matching the prespacer length. Cas9 dissociation triggers structural reconfiguration of the Csn2-Cas1-Cas2 assembly. This exposes the PAM-proximal DNA, allowing Cas1-Cas2 to bind the exposed site for subsequent prespacer processing and directional integration. These findings reveal how Cas9, Csn2, and Cas1-Cas2 couple PAM recognition with prespacer selection, ensuring fidelity during adaptation.
History
DepositionJun 19, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 1, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 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: Type II-A CRISPR-associated protein Csn2
B: Type II-A CRISPR-associated protein Csn2
C: Type II-A CRISPR-associated protein Csn2
D: Type II-A CRISPR-associated protein Csn2
I: CRISPR-associated endonuclease Cas1
J: CRISPR-associated endonuclease Cas1
X: DNA (51-MER)
Y: DNA (51-MER)
N: CRISPR-associated endoribonuclease Cas2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,33315
Polymers213,0929
Non-polymers2406
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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CRISPR-associated ... , 2 types, 3 molecules IJN

#2: Protein CRISPR-associated endonuclease Cas1


Mass: 33492.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus faecalis (bacteria) / Gene: cas1, EGW70_05065, EY666_02190 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A3N3GDE4, Hydrolases; Acting on ester bonds
#5: Protein CRISPR-associated endoribonuclease Cas2


Mass: 12977.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus faecalis (bacteria) / Gene: cas2, EGW70_05060, EY666_02195, GTI81_07750 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A3N3FLR9, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 2 molecules XY

#3: DNA chain DNA (51-MER)


Mass: 15468.875 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (51-MER)


Mass: 15928.584 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Non-polymers , 2 types, 10 molecules ABCD

#1: Protein
Type II-A CRISPR-associated protein Csn2


Mass: 25433.090 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus faecalis (bacteria) / Gene: csn2, EGW70_05055, EY666_02200, GTI81_07755 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3N3GYE7
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestN
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: E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1
Type: COMPLEX / Entity ID: #1-#2, #5, #3-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Enterococcus faecalis (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 magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13PHENIX3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50477 / Symmetry type: POINT
RefinementHighest resolution: 3.76 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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