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- EMDB-65109: Type II-A CRISPR integrase pre-integration complex -

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Basic information

Entry
Database: EMDB / ID: EMD-65109
TitleType II-A CRISPR integrase pre-integration complex
Map data
Sample
  • Complex: E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1
    • Protein or peptide: Type II-A CRISPR-associated protein Csn2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • DNA: DNA (51-MER)
    • DNA: DNA (51-MER)
  • Ligand: CALCIUM ION
KeywordsCRISPR / Cas1 / Cas2 / Csn2 / DNA BINDING PROTEIN/DNA / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsLi ZX / Li YT / Lu ML / Xiao YB
Funding support China, 3 items
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, 2025-
Header (metadata) releaseApr 1, 2026-
Map releaseApr 1, 2026-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65109.png

Downloads & links

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Map

FileDownload / File: emd_65109.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 256 pix.
= 238.592 Å		0.93 Å/pix.
x 256 pix.
= 238.592 Å		0.93 Å/pix.
x 256 pix.
= 238.592 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.932 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.18499611 - 0.3998499
Average (Standard dev.)0.0021691923 (±0.01677334)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 238.592 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_65109_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_65109_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_65109_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1

EntireName: E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1
Components
  • Complex: E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1
    • Protein or peptide: Type II-A CRISPR-associated protein Csn2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • DNA: DNA (51-MER)
    • DNA: DNA (51-MER)
  • Ligand: CALCIUM ION

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Supramolecule #1: E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1

SupramoleculeName: E. faecalis Csn2/Cas1/Cas2 pre-integration complex, conformation 1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #5, #3-#4
Source (natural)Organism: Enterococcus faecalis (bacteria)

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

MacromoleculeName: Type II-A CRISPR-associated protein Csn2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus faecalis (bacteria)
Molecular weightTheoretical: 25.43309 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRVNFSLLEE PIEIEKATFL TIKDVQTFAH LVKLIYQYDG ENELKLFDAQ QKGLKPTELF VVTDILGYDV NSAATLKLIY GDLEAQLND KPEVKSMIEK LTGTISQLIG YELLEHEMDL EEDGITVQEL FKALGIKIET TSDTIFEKVM EITQVHRYLS K KKLLIFIN ...String:
MRVNFSLLEE PIEIEKATFL TIKDVQTFAH LVKLIYQYDG ENELKLFDAQ QKGLKPTELF VVTDILGYDV NSAATLKLIY GDLEAQLND KPEVKSMIEK LTGTISQLIG YELLEHEMDL EEDGITVQEL FKALGIKIET TSDTIFEKVM EITQVHRYLS K KKLLIFIN ACTYLTEDEV QQVVEYISLN NVDVLFLEQR VVQNRFQYIL DENFYLSYEK A

UniProtKB: Type II-A CRISPR-associated protein Csn2

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Macromolecule #2: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Enterococcus faecalis (bacteria)
Molecular weightTheoretical: 33.492625 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGWRTVVVNK HSKLSYKNNH LVFKAIDHQE LIHLSEIDVL LLETTDISLT TMLLKRLIDE KILVLFCDDK RLPIGKILPF YGRHDSSLQ LTRQLAWTEE RKGQVWTAII AQKITNQSLH LAQRDYGQKA AALLAMRAEL RLFDPANREG HAARSYFNTL F GNDFTREQ ...String:
MGWRTVVVNK HSKLSYKNNH LVFKAIDHQE LIHLSEIDVL LLETTDISLT TMLLKRLIDE KILVLFCDDK RLPIGKILPF YGRHDSSLQ LTRQLAWTEE RKGQVWTAII AQKITNQSLH LAQRDYGQKA AALLAMRAEL RLFDPANREG HAARSYFNTL F GNDFTREQ ENDINAGLNY GYTLLLSIFA RELVQTGCFT QLGLKHANQF NDFNLASDLM EPFRPLVDQI IYENRKEAFP IM KRKLFAL FMNTYMYKKK QMFLTNIATD YTKHVVKVLN QEEEGVPEFG I

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #5: CRISPR-associated endoribonuclease Cas2

MacromoleculeName: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Enterococcus faecalis (bacteria)
Molecular weightTheoretical: 12.977047 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSYRYMRLLL MFDMPTDTAS DRKAYRKFRK FLINEGFIMH QFSVYSKILL NDTANKAMLA RLKQNNPQRG LITLLNVTEK QFSRMIYLH GEQDNRVANS DERIVFLGEE

UniProtKB: CRISPR-associated endoribonuclease Cas2

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Macromolecule #3: DNA (51-MER)

MacromoleculeName: DNA (51-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.468875 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)

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Macromolecule #4: DNA (51-MER)

MacromoleculeName: DNA (51-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.928584 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / 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: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX / Number images used: 50477
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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