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- EMDB-17722: Cas1-Cas2 CRISPR integrase bound to prespacer DNA, Streptococcus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-17722
TitleCas1-Cas2 CRISPR integrase bound to prespacer DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Map dataMap autosharpened using phenix.auto_sharpen_1.21rc1-4903 b_iso_to_d_cut and 3.17 A cutoff
Sample
  • Complex: Cas1-Cas2 CRISPR integrase-prespacer DNA complex
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • DNA: Chains: G,J
    • DNA: Chains: H,K
  • Ligand: MAGNESIUM 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: 3.17 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
DepositionJun 23, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17722.png

Downloads & links

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Map

FileDownload / File: emd_17722.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 b_iso_to_d_cut and 3.17 A cutoff
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: 5.0
Minimum - Maximum-47.108359999999998 - 54.809649999999998
Average (Standard dev.)0.000000000001833 (±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_17722_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_17722_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17722_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17722_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-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: Chains: G,J
    • DNA: Chains: H,K
  • Ligand: MAGNESIUM 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: 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: Chains: G,J

MacromoleculeName: Chains: G,J / 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: Chains: H,K

MacromoleculeName: Chains: H,K / 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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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
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 / 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: 3 / Number real images: 3373 / 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
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91658
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-8pk1:
Cas1-Cas2 CRISPR integrase bound to prespacer DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system

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