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- EMDB-51766: Cas1-Cas2 CRISPR integrase bound to prespacer DNA, Streptococcus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-51766
TitleCas1-Cas2 CRISPR integrase bound to prespacer DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Map dataSharpened map (phenix.auto_sharpen)
Sample
  • Complex: Cas1-Cas2 CRISPR integrase-prespacer DNA complex
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • DNA: Prespacer DNA (26-MER)
    • DNA: Prespacer DNA (26-MER)
  • Ligand: CALCIUM ION
KeywordsCas1-Cas2 integrase / CRISPR-Cas / prespacer / spacer acquisition / DNA BINDING PROTEIN
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 / DNA binding / metal ion binding
Similarity search - Function
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 endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2
Similarity search - Component
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 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
DepositionOct 9, 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_51766.png

Downloads & links

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Map

FileDownload / File: emd_51766.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map (phenix.auto_sharpen)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 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: 5.0
Minimum - Maximum-38.090584 - 61.593772999999999
Average (Standard dev.)-0.00000000000621 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51766_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map. Input for phenix.autosharpen (b iso to d cut 3.06A resolution)...

Fileemd_51766_additional_1.map
Annotationunsharpened map. Input for phenix.autosharpen (b_iso_to_d_cut 3.06A resolution)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_51766_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_51766_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas1-Cas2 CRISPR integrase-prespacer DNA complex

EntireName: Cas1-Cas2 CRISPR integrase-prespacer DNA complex
Components
  • Complex: Cas1-Cas2 CRISPR integrase-prespacer DNA complex
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • DNA: Prespacer DNA (26-MER)
    • DNA: Prespacer DNA (26-MER)
  • Ligand: CALCIUM ION

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Supramolecule #1: Cas1-Cas2 CRISPR integrase-prespacer DNA complex

SupramoleculeName: Cas1-Cas2 CRISPR integrase-prespacer DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)

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Macromolecule #1: CRISPR-associated endoribonuclease Cas2

MacromoleculeName: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 13.43156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSYRYMRMIL MFDMPTDTAE ERKAYRKFRK FLLSEGFIMH QFSVYSKLLL NHTANTAMVG RLKANNPKKG NITILTVTEK QFARMIYLY GDKNTSIANS EERLVFLGDN YCDED

UniProtKB: CRISPR-associated endoribonuclease Cas2

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Macromolecule #2: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 33.863836 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGWRTVVVN IHSKLSYKNN HLIFRNSYKT EMIHLSEIDI LLLETTDIVL TTMLVKRLVD ENILVIFCDD KRLPTAFLTP YYARHDSSL QIARQIAWKE NVKCEVWTAI IAQKILNQSY YLGECSFFEK SQSIMELYHG LERFDPSNRE GHSARIYFNT L FGNDFTRE ...String:
MAGWRTVVVN IHSKLSYKNN HLIFRNSYKT EMIHLSEIDI LLLETTDIVL TTMLVKRLVD ENILVIFCDD KRLPTAFLTP YYARHDSSL QIARQIAWKE NVKCEVWTAI IAQKILNQSY YLGECSFFEK SQSIMELYHG LERFDPSNRE GHSARIYFNT L FGNDFTRE SDNDINAALD YGYTLLLSMF AREVVVCGCM TQIGLKHANQ FNQFNLASDI MEPFRPIIDR IVYQNRHNNF VK IKKELFS IFSETYLYNG KEMYLSNIVS DYTKKVIKAL NQLGEEIPEF RI

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #3: Prespacer DNA (26-MER)

MacromoleculeName: Prespacer DNA (26-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.917082 KDa
SequenceString:
(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DC)(DT)(DC) (DG)(DT)(DT)(DC)(DT)(DG)(DG)(DT)(DG)(DT) (DT)(DT)(DC)(DT)(DC)(DG)

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Macromolecule #4: Prespacer DNA (26-MER)

MacromoleculeName: Prespacer DNA (26-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.062258 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DA)(DC)(DC)(DA)(DG)(DA) (DA)(DC)(DG)(DA)(DG)(DT)(DA)(DG)(DT)(DA) (DA)(DA)(DT)(DT)(DG)(DG)

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 2 / 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
Component:
ConcentrationFormulaName
50.0 mMNaClsodium chloride
2.0 mMCaCl2calcium chloride
5.0 mMTris-HCl pH 7.5
1.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE OXIDE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / 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 / Number grids imaged: 3 / Number real images: 3404 / Average exposure time: 46.33 sec. / Average electron dose: 30.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / 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

Particle selectionDetails: Blob picking in cryoSPARC live session
CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.0) / Number images used: 136273
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-9h1h:
Cas1-Cas2 CRISPR integrase bound to prespacer DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system

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