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- PDB-9p11: Cas1-Cas2/3 integrase, heterohexameric assembly -

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Basic information

Entry
Database: PDB / ID: 9p11
TitleCas1-Cas2/3 integrase, heterohexameric assembly
Components
  • CRISPR-associated endonuclease Cas1
  • CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
KeywordsRECOMBINATION / CRISPR / integrase / type I-F
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / DNA endonuclease activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / DNA binding / ATP binding / metal ion binding / identical protein binding
Similarity search - Function
Helicase Cas3, CRISPR-associated, Yersinia-type / : / : / CRISPR-associated nuclease/helicase Cas3, I-F/YPEST, Cas2 domain / CRISPR-associated Cas3 subtype I-F/YPEST-like, C-terminal domain / CRISPR-associated protein Cas1, YPEST subtype / Cas3, HD domain / : / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily ...Helicase Cas3, CRISPR-associated, Yersinia-type / : / : / CRISPR-associated nuclease/helicase Cas3, I-F/YPEST, Cas2 domain / CRISPR-associated Cas3 subtype I-F/YPEST-like, C-terminal domain / CRISPR-associated protein Cas1, YPEST subtype / Cas3, HD domain / : / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / CRISPR-associated nuclease/helicase Cas3, C-terminal / HD Cas3-type domain profile. / CRISPR-associated endonuclease Cas1, N-terminal domain / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas1 / CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHenriques, W.S. / Bowman, J. / Hall, L.N. / Gauvin, C.G. / Wei, H. / Kuang, H. / Zimanyi, C.M. / Eng, E.T. / Santiago-Frangos, A. / Wiedenheft, B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 United States
CitationJournal: Structure / Year: 2025
Title: Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.
Authors: William S Henriques / Jarrett Bowman / Laina N Hall / Colin C Gauvin / Hui Wei / Huihui Kuang / Christina M Zimanyi / Edward T Eng / Andrew Santiago-Frangos / Blake Wiedenheft /
Abstract: Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain ...Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.
History
DepositionJun 9, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: CRISPR-associated endonuclease Cas1
D: CRISPR-associated endonuclease Cas1
A: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
B: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
C: CRISPR-associated endonuclease Cas1
E: CRISPR-associated endonuclease Cas1


Theoretical massNumber of molelcules
Total (without water)387,1676
Polymers387,1676
Non-polymers00
Water00
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 Cas1


Mass: 36154.844 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: cas1, PA14_33350 / Production host: Escherichia coli (E. coli)
References: UniProt: Q02ML7, Hydrolases; Acting on ester bonds
#2: Protein CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST


Mass: 121273.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: cas3, PA14_33340 / Production host: Escherichia coli (E. coli)
References: UniProt: Q02ML8, Hydrolases; Acting on ester bonds, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
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: Cas1-2/3 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.380 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse peak on SEC
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingAverage exposure time: 2 sec. / Electron dose: 52.51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3944

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1716883 / Details: cryoSPARC template picker
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 303250 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 51.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002825002
ELECTRON MICROSCOPYf_angle_d0.488734066
ELECTRON MICROSCOPYf_chiral_restr0.03643866
ELECTRON MICROSCOPYf_plane_restr0.0044450
ELECTRON MICROSCOPYf_dihedral_angle_d3.59813662

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