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- EMDB-71115: Cas1-2/3 integration complex, 32 bp foreign DNA without PAM, firs... -

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Basic information

Entry
Database: EMDB / ID: EMD-71115
TitleCas1-2/3 integration complex, 32 bp foreign DNA without PAM, first transesterification
Map data
Sample
  • Complex: Integration complex formed by Cas1-2/3 integrase with integration host factor alpha and beta, the CRISPR leader and CRISPR array, and a short foreign DNA fragment
    • Complex: Cas1-2/3 integrase and associated proteins
    • Complex: Nucleic Acid
KeywordsCRISPR / integrase / type I-F / RECOMBINATION
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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 10, 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_71115.png

Downloads & links

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Map

FileDownload / File: emd_71115.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 324 pix.
= 357.696 Å		1.1 Å/pix.
x 324 pix.
= 357.696 Å		1.1 Å/pix.
x 324 pix.
= 357.696 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.104 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.103
Minimum - Maximum-0.27780932 - 0.58873594
Average (Standard dev.)-0.00001139133 (±0.016156664)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 357.69598 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_71115_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_71115_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Integration complex formed by Cas1-2/3 integrase with integration...

EntireName: Integration complex formed by Cas1-2/3 integrase with integration host factor alpha and beta, the CRISPR leader and CRISPR array, and a short foreign DNA fragment
Components
  • Complex: Integration complex formed by Cas1-2/3 integrase with integration host factor alpha and beta, the CRISPR leader and CRISPR array, and a short foreign DNA fragment
    • Complex: Cas1-2/3 integrase and associated proteins
    • Complex: Nucleic Acid

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Supramolecule #1: Integration complex formed by Cas1-2/3 integrase with integration...

SupramoleculeName: Integration complex formed by Cas1-2/3 integrase with integration host factor alpha and beta, the CRISPR leader and CRISPR array, and a short foreign DNA fragment
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: Cas1-2/3 integrase and associated proteins

SupramoleculeName: Cas1-2/3 integrase and associated proteins / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #3: Nucleic Acid

SupramoleculeName: Nucleic Acid / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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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

BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 5462 / Average electron dose: 65.67 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 36000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 131056
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: COMMON LINE
FSC plot (resolution estimation)

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