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- EMDB-65107: Raw consensus map of Type II-A CRISPR integrase prespacer catchin... -

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Basic information

Entry
Database: EMDB / ID: EMD-65107
TitleRaw consensus map of Type II-A CRISPR integrase prespacer catching complex, State I
Map data
Sample
  • Complex: E. faecalis Type II-A CRISPR integrase prespacer catching complex, State I
    • DNA: DNA (51-MER)
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • DNA: DNA (51-MER)
    • RNA: RNA (55-MER)
    • RNA: RNA (83-MER)
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: Type II-A CRISPR-associated protein Csn2
KeywordsCRISPR / Cas9 / Cas1 / Csn2 DNA binding protein / DNA BINDING PROTEIN/DNA/RNA / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / DNA endonuclease activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / DNA binding / RNA 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 protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe ...CRISPR-associated protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas1 / Type II-A CRISPR-associated protein Csn2 / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesEnterococcus faecalis (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.96 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_65107.png

Downloads & links

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Map

FileDownload / File: emd_65107.map.gz / Format: CCP4 / Size: 216 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 384 pix.
= 357.888 Å		0.93 Å/pix.
x 384 pix.
= 357.888 Å		0.93 Å/pix.
x 384 pix.
= 357.888 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: 0.143
Minimum - Maximum-0.0043593664 - 2.0358155
Average (Standard dev.)0.00094085623 (±0.020782616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 357.888 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_65107_additional_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #2

Fileemd_65107_additional_3.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_65107_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_65107_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : E. faecalis Type II-A CRISPR integrase prespacer catching complex...

EntireName: E. faecalis Type II-A CRISPR integrase prespacer catching complex, State I
Components
  • Complex: E. faecalis Type II-A CRISPR integrase prespacer catching complex, State I
    • DNA: DNA (51-MER)
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • DNA: DNA (51-MER)
    • RNA: RNA (55-MER)
    • RNA: RNA (83-MER)
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: Type II-A CRISPR-associated protein Csn2

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Supramolecule #1: E. faecalis Type II-A CRISPR integrase prespacer catching complex...

SupramoleculeName: E. faecalis Type II-A CRISPR integrase prespacer catching complex, State I
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3, #7, #4-#6, #2, #1
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 #7: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Enterococcus faecalis (bacteria)
Molecular weightTheoretical: 155.565297 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKDYVIGLD IGTNSVGWAV MTEDYQLVKK KMPIYGNTEK KKIKKNFWGV RLFEEGHTAE DRRLKRTARR RISRRRNRLR YLQAFFEEA MTDLDENFFA RLQESFLVPE DKKWHRHPIF AKLEDEVAYH ETYPTIYHLR KKLADSSEQA DLRLIYLALA H IVKYRGHF ...String:
MKKDYVIGLD IGTNSVGWAV MTEDYQLVKK KMPIYGNTEK KKIKKNFWGV RLFEEGHTAE DRRLKRTARR RISRRRNRLR YLQAFFEEA MTDLDENFFA RLQESFLVPE DKKWHRHPIF AKLEDEVAYH ETYPTIYHLR KKLADSSEQA DLRLIYLALA H IVKYRGHF LIEGKLSTEN ISVKEQFQQF MIIYNQTFVN GESRLVSAPL PESVLIEEEL TEKASRTKKS EKVLQQFPQE KA NGLFGQF LKLMVGNKAD FKKVFGLEEE AKITYASESY EEDLEGILAK VGDEYSDVFL AAKNVYDAVE LSTILADSDK KSH AKLSSS MIVRFTEHQE DLKKFKRFIR ENCPDEYDNL FKNEQKDGYA GYIAHAGKVS QLKFYQYVKK IIQDIAGAEY FLEK IAQEN FLRKQRTFDN GVIPHQIHLA ELQAIIHRQA AYYPFLKENQ EKIEQLVTFR IPYYVGPLSK GDASTFAWLK RQSEE PIRP WNLQETVDLD QSATAFIERM TNFDTYLPSE KVLPKHSLLY EKFMVFNELT KISYTDDRGI KANFSGKEKE KIFDYL FKT RRKVKKKDII QFYRNEYNTE IVTLSGLEED QFNASFSTYQ DLLKCGLTRA ELDHPDNAEK LEDIIKILTI FEDRQRI RT QLSTFKGQFS AEVLKKLERK HYTGWGRLSK KLINGIYDKE SGKTILGYLI KDDGVSKHYN RNFMQLINDS QLSFKNAI Q KAQSSEHEET LSETVNELAG SPAIKKGIYQ SLKIVDELVA IMGYAPKRIV VEMARENQTT STGKRRSIQR LKIVEKAMA EIGSNLLKEQ PTTNEQLRDT RLFLYYMQNG KDMYTGDELS LHRLSHYDID HIIPQSFMKD DSLDNLVLVG STENRGKSDD VPSKEVVKD MKAYWEKLYA AGLISQRKFQ RLTKGEQGGL TLEDKAHFIQ RQLVETRQIT KNVAGILDQR YNANSKEKKV Q IITLKASL TSQFRSIFGL YKVREVNDYH HGQDAYLNCV VATTLLKVYP NLAPEFVYGE YPKFQTFKEN KATAKAIIYT NL LRFFTED EPRFTKDGEI LWSNSYLKTI KKELNYHQMN IVKKVEVQKG GFSKESIKPK GPSNKLIPVK NGLDPQKYGG FDS PIVAYT VLFTHEKGKK PLIKQEILGI TIMEKTRFEQ NPILFLEEKG FLRPRVLMKL PKYTLYEFPE GRRRLLASAK EAQK GNQMV LPEHLLTLLY HAKQCLLPNQ SESLAYVEQH QPEFQEILER VVDFAEVHTL AKSKVQQIVK LFEANQTADV KEIAA SFIQ LMQFNAMGAP STFKFFQKDI ERARYTSIKE IFDATIIYQS TTGLYETRRK VVD

UniProtKB: CRISPR-associated endonuclease Cas9

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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: 15.774186 KDa
SequenceString:
(DA)(DG)(DC)(DG)(DC)(DT)(DC)(DT)(DG)(DA) (DA)(DT)(DG)(DC)(DT)(DA)(DG)(DA)(DT)(DC) (DA)(DC)(DG)(DT)(DT)(DA)(DT)(DG)(DA) (DA)(DA)(DC)(DA)(DG)(DA)(DC)(DA)(DT)(DA) (DG) (DC)(DG)(DG)(DT)(DA)(DC)(DA)(DA) (DA)(DA)(DA)

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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: 15.644021 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DG)(DT)(DA)(DC)(DC) (DG)(DC)(DT)(DA)(DT)(DG)(DT)(DC)(DT)(DG) (DT)(DT)(DT)(DC)(DA)(DT)(DA)(DA)(DC) (DG)(DT)(DG)(DA)(DT)(DC)(DT)(DA)(DG)(DC) (DA) (DT)(DT)(DC)(DA)(DG)(DA)(DG)(DC) (DG)(DC)(DT)

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Macromolecule #5: RNA (55-MER)

MacromoleculeName: RNA (55-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.561324 KDa
SequenceString:
GCAAUACUUU GUCUGUAUCG UUUUAGAGUC AUGUUGUUUA GAAUGGUACC AAAAC

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Macromolecule #6: RNA (83-MER)

MacromoleculeName: RNA (83-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 26.614762 KDa
SequenceString:
CUUUUGGGAC UAUUCUAAAC AACAUAGCAA GUUAAAAUAA GGUUUUAACC GUAAUCAACU GUAAAGUGGC GCUGUUUCGG CGC

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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: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 136878
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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