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- EMDB-65232: Focused map of Type II-A CRISPR integrase prespacer catching comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-65232
TitleFocused map of Type II-A CRISPR integrase prespacer catching complex, State II
Map data
Sample
  • Complex: Type II-A CRISPR integrase prespacer catching complex
KeywordsCRISPR / Cas9 / Cas1 / Csn2 DNA binding protein / DNA BINDING PROTEIN/DNA/RNA / DNA BINDING PROTEIN-DNA-RNA complex / DNA BINDING PROTEIN
Biological speciesEnterococcus faecalis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsLi ZX / Xiao YB
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970547 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
DepositionJul 2, 2025-
Header (metadata) releaseMay 13, 2026-
Map releaseMay 13, 2026-
UpdateMay 13, 2026-
Current statusMay 13, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65232.png

Downloads & links

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Map

FileDownload / File: emd_65232.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.932 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.3387586 - 0.6983891
Average (Standard dev.)-0.00019645259 (±0.013100918)
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_65232_additional_1.map
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Additional map: #2

Fileemd_65232_additional_2.map
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Additional map: #3

Fileemd_65232_additional_3.map
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Half map: #1

Fileemd_65232_half_map_1.map
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Half map: #2

Fileemd_65232_half_map_2.map
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Sample components

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Entire : Type II-A CRISPR integrase prespacer catching complex

EntireName: Type II-A CRISPR integrase prespacer catching complex
Components
  • Complex: Type II-A CRISPR integrase prespacer catching complex

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

SupramoleculeName: Type II-A CRISPR integrase prespacer catching complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Enterococcus faecalis (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
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: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 80591
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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