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- EMDB-52321: Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound confor... -

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Basic information

Entry
Database: EMDB / ID: EMD-52321
TitleCas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound conformation, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Map dataUnsharpened consensus map
Sample
  • Complex: Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound conformation
KeywordsCIRSPR-Cas / spacer acquisition / Csn2 / Cas1 / Cas2 / CRISPR integrase / DNA BINDING PROTEIN
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsSasnauskas G / Gaizauskaite U / Tamulaitiene G
Funding supportLithuania, 1 items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-19-32Lithuania
CitationJournal: Mol Cell / Year: 2026
Title: Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation.
Authors: Ugne Gaizauskaite / Giedre Tamulaitiene / Arunas Silanskas / Giedrius Gasiunas / Virginijus Siksnys / Giedrius Sasnauskas
Abstract: During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer ...During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems, selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a "supercomplex" with the Cas1-Cas2 integrase and the Csn2 protein. Here, we present cryo-electron microscopy structures of the Streptococcus thermophilus type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing Cas9 by the CRISPR adaptation machinery for prespacer selection, as characterized here, demonstrates Cas9 plasticity and expands our knowledge of Cas9 biology.
History
DepositionDec 12, 2024-
Header (metadata) releaseFeb 18, 2026-
Map releaseFeb 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52321.png

Downloads & links

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Map

FileDownload / File: emd_52321.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened consensus map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 336 pix.
= 369.6 Å		1.1 Å/pix.
x 336 pix.
= 369.6 Å		1.1 Å/pix.
x 336 pix.
= 369.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.4755546 - 1.8391184
Average (Standard dev.)0.00086874043 (±0.053458318)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 369.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52321_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_52321_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_52321_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound confor...

EntireName: Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound conformation
Components
  • Complex: Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound conformation

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Supramolecule #1: Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound confor...

SupramoleculeName: Cas9-Cas1-Cas2-Csn2 supercomplex with dsDNA in PAM-unbound conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (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 R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: Glowcube plus, 20 mA
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average exposure time: 46.33 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: cryoSPARC ab-initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 67204
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9h2m


Chain - Source name: PDB / Chain - Initial model type: experimental model

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