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- EMDB-18113: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Str... -

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Basic information

Entry
Database: EMDB / ID: EMD-18113
TitleCas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Map dataMap autosharpened using phenix.auto_sharpen_1.21rc1-4903 and b_iso_to_d_cut
Sample
  • Complex: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • DNA: Prespacer DNA, chain G
    • DNA: Prespacer DNA, chain H
    • DNA: Integration target, chain I
    • DNA: Integration target, chain J
  • Ligand: MAGNESIUM ION
KeywordsCas1-Cas2 integrase / CRISPR-Cas / prespacer / CRISPR leader / CRISPR repeat / 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: 2.98 Å
AuthorsSasnauskas G / Gaizauskaite U / Tamulaitiene G
Funding supportLithuania, 1 items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-19-32Lithuania
CitationJournal: To be published
Title: Structural basis for spacer acquisition in a type II-A CRISPR-Cas system
Authors: Sasnauskas G / Gaizauskaite U / Tamulaitiene G
History
DepositionAug 2, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18113.png

Downloads & links

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Map

FileDownload / File: emd_18113.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap autosharpened using phenix.auto_sharpen_1.21rc1-4903 and b_iso_to_d_cut
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. 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: 6.0
Minimum - Maximum-25.895664 - 51.043835000000001
Average (Standard dev.)-0.000000000003314 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18113_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened main map

Fileemd_18113_additional_1.map
AnnotationUnsharpened main map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18113_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_18113_half_map_2.map
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 bound to prespacer and target DNA, Str...

EntireName: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Components
  • Complex: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • DNA: Prespacer DNA, chain G
    • DNA: Prespacer DNA, chain H
    • DNA: Integration target, chain I
    • DNA: Integration target, chain J
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Str...

SupramoleculeName: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
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: 35.363461 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 RILESGWSHP QFEKA

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #3: Prespacer DNA, chain G

MacromoleculeName: Prespacer DNA, chain G / type: dna / ID: 3 / Number of copies: 1 / 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 #4: Prespacer DNA, chain H

MacromoleculeName: Prespacer DNA, chain H / type: dna / ID: 4 / Number of copies: 1 / 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 #5: Integration target, chain I

MacromoleculeName: Integration target, chain I / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.729506 KDa
SequenceString:
(DT)(DA)(DC)(DG)(DA)(DG)(DG)(DT)(DT)(DT) (DT)(DG)(DG)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DC)(DG)(DA)(DA)(DA)(DC)(DA)(DA)(DC) (DA)(DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA)(DA) (DA) (DA)(DC)(DC)(DT)(DC)(DG)(DT)(DA)

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Macromolecule #6: Integration target, chain J

MacromoleculeName: Integration target, chain J / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.835518 KDa
SequenceString:
(DT)(DA)(DC)(DG)(DA)(DG)(DG)(DT)(DT)(DT) (DT)(DA)(DG)(DA)(DG)(DC)(DT)(DG)(DT)(DG) (DT)(DT)(DG)(DT)(DT)(DT)(DC)(DG)(DA) (DA)(DT)(DG)(DG)(DT)(DT)(DC)(DC)(DA)(DA) (DA) (DA)(DC)(DC)(DT)(DC)(DG)(DT)(DA)

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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
100.0 mMNaCl
2.0 mMCa(OAc)2calcium acetate
10.0 mMTris-HCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
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: 1 / Number real images: 3129 / Average exposure time: 46.33 sec. / Average electron dose: 29.7 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination 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 selectionNumber selected: 1557793 / Details: blob picking in cryoSPARC live session
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: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1+230427) / Details: Non-uniform refinement in cryoSPARC / Number images used: 82944
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-8q2n:
Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system

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