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- EMDB-71097: Cas1-Cas2/3 integrase bound to foreign dsDNA fragment -

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Basic information

Entry
Database: EMDB / ID: EMD-71097
TitleCas1-Cas2/3 integrase bound to foreign dsDNA fragment
Map data
Sample
  • Complex: Cas1-2/3 with bound foreign dsDNA
    • Complex: Cas1-2/3
      • Protein or peptide: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
      • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Complex: dsDNA
      • DNA: DNA Strand 1
      • DNA: DNA Strand 2
KeywordsCRISPR / integrase / type I-F / RECOMBINATION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsHenriques WS / Bowman J / Hall LN / Gauvin CG / Wei H / Kuang H / Zimanyi CM / Eng ET / Santiago-Frangos A / Wiedenheft B
Funding support United States, 1 items
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, 2025-
Header (metadata) releaseOct 22, 2025-
Map releaseOct 22, 2025-
UpdateOct 22, 2025-
Current statusOct 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71097.png

Downloads & links

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Map

FileDownload / File: emd_71097.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 333.2 Å		0.83 Å/pix.
x 400 pix.
= 333.2 Å		0.83 Å/pix.
x 400 pix.
= 333.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.178
Minimum - Maximum-0.55606955 - 1.1100007
Average (Standard dev.)0.0002923546 (±0.030371787)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 333.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_71097_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_71097_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas1-2/3 with bound foreign dsDNA

EntireName: Cas1-2/3 with bound foreign dsDNA
Components
  • Complex: Cas1-2/3 with bound foreign dsDNA
    • Complex: Cas1-2/3
      • Protein or peptide: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
      • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Complex: dsDNA
      • DNA: DNA Strand 1
      • DNA: DNA Strand 2

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Supramolecule #1: Cas1-2/3 with bound foreign dsDNA

SupramoleculeName: Cas1-2/3 with bound foreign dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 415 KDa

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Supramolecule #2: Cas1-2/3

SupramoleculeName: Cas1-2/3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #3: dsDNA

SupramoleculeName: dsDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Synthetically produced: Yes

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Macromolecule #1: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST

MacromoleculeName: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 121.27368 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNILLVSQCE KRALSETRRI LDQFAERRGE RTWQTPITQA GLDTLRRLLK KSARRNTAVA CHWIRGRDHS ELLWIVGDAS RFNAQGAVP TNRTCRDILR KEDENDWHSA EDIRLLTVMA ALFHDIGKAS QAFQAKLRNR GKPMADAYRH EWVSLRLFEA F VGPGSSDE ...String:
MNILLVSQCE KRALSETRRI LDQFAERRGE RTWQTPITQA GLDTLRRLLK KSARRNTAVA CHWIRGRDHS ELLWIVGDAS RFNAQGAVP TNRTCRDILR KEDENDWHSA EDIRLLTVMA ALFHDIGKAS QAFQAKLRNR GKPMADAYRH EWVSLRLFEA F VGPGSSDE DWLRRLADKR ETGDAWLSQL ARDDRQSAPP GPFQKSRLPP LAQAVGWLIV SHHRLPNGDH RGSASLARLP AP IQSQWCG ARDADAKEKA ACWQFPHGLP FASAHWRART ALCAQSMLER PGLLARGPAL LHDSYVMHVS RLILMLADHH YSS LPADSR LGDPNFPLHA NTDRDSGKLK QRLDEHLLGV ALHSRKLAGT LPRLERQLPR LARHKGFTRR VEQPRFRWQD KAYD CAMAC REQAMEHGFF GLNLASTGCG KTLANGRILY ALADPQRGAR FSIALGLRSL TLQTGQAYRE RLGLGDDDLA ILVGG SAAR ELFEKQQERL ERSGSESAQE LLAENSHVHF AGTLEDGPLR EWLGRNSAGN RLLQAPILAC TIDHLMPASE SLRGGH QIA PLLRLMTSDL VLDEVDDFDI DDLPALSRLV HWAGLFGSRV LLSSATLPPA LVQGLFEAYR SGREIFQRHR GAPGRAT EI RCAWFDEFSS QSSAHGAVTS FSEAHATFVA QRLAKLEQLP PRRQAQLCTV HAAGEARPAL CRELAGQMNT WMADLHRC H HTEHQGRRIS FGLLRLANIE PLIELAQAIL AQGAPEGLHV HLCVYHSRHP LLVRSAIERQ LDELLKRSDD DAAALFARP TLAKALQAST ERDHLFVVLA SPVAEVGRDH DYDWAIVEPS SMRSIIQLAG RIRRHRSGFS GEANLYLLSR NIRSLEGQNP AFQRPGFET PDFPLDSHDL HDLLDPALLA RIDASPRIVE PFPLFPRSRL VDLEHRRLRA LMLADDPPSS LLGVPLWWQT P ASLSGALQ TSQPFRAGAK ERCYALLPDE DDEERLHFSR YEEGTWSNQD NLLRNLDLTY GPRIQTWGTV NYREELVAMA GR EDLDLRQ CAMRYGEVRL RENTQGWSYH PYLGFKKYN

UniProtKB: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST

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Macromolecule #2: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 2 / Details: Strep-tagged Cas1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 36.154844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDDISPSELK TILHSKRANL YYLQHCRVLV NGGRVEYVTD EGRHSHYWNI PIANTTSLLL GTGTSITQAA MRELARAGVL VGFCGGGGT PLFSANEVDV EVSWLTPQSE YRPTEYLQRW VGFWFDEEKR LVAARHFQRA RLERIRHSWL EDRVLRDAGF A VDATALAV ...String:
MDDISPSELK TILHSKRANL YYLQHCRVLV NGGRVEYVTD EGRHSHYWNI PIANTTSLLL GTGTSITQAA MRELARAGVL VGFCGGGGT PLFSANEVDV EVSWLTPQSE YRPTEYLQRW VGFWFDEEKR LVAARHFQRA RLERIRHSWL EDRVLRDAGF A VDATALAV AVEDSARALE QAPNHEHLLT EEARLSKRLF KLAAQATRYG EFVRAKRGSG GDPANRFLDH GNYLAYGLAA TA TWVLGIP HGLAVLHGKT RRGGLVFDVA DLIKDSLILP QAFLSAMRGD EEQDFRQACL DNLSRAQALD FMIDTLKDVA QRS TVSA

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #3: DNA Strand 1

MacromoleculeName: DNA Strand 1 / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 10.377702 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DC)(DC)(DT)(DG)(DG) (DA)(DC)(DT)(DA)(DC)(DT)(DA)(DC)(DA)(DA) (DC)(DC)(DT)(DT)(DC)(DG)(DC)(DT)(DT) (DT)(DT)(DT)(DG)(DG)

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Macromolecule #4: DNA Strand 2

MacromoleculeName: DNA Strand 2 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 9.959435 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DG)(DC)(DG)(DA)(DA) (DG)(DG)(DT)(DT)(DG)(DT)(DA)(DG)(DT)(DA) (DG)(DT)(DC)(DC)(DA)(DG)(DG)(DA)(DA) (DA)(DA)(DA)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.7 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsMonodisperse peak on SEC

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 5552 / Average exposure time: 6.0 sec. / Average electron dose: 51.78 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 96000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1083100 / Details: crYolo
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 28665
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: COMMON LINE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainPDB ID
source_name: AlphaFold, initial_model_type: in silico model
source_name: PDB, initial_model_type: experimental model

8flj

RefinementProtocol: RIGID BODY FIT
Output model

PDB-9p1d:
Cas1-Cas2/3 integrase bound to foreign dsDNA fragment

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