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- PDB-8q2n: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Str... -

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Basic information

Entry
Database: PDB / ID: 8q2n
TitleCas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Components
  • (CRISPR-associated ...) x 2
  • (Integration target, chain ...) x 2
  • (Prespacer DNA, chain ...) x 2
KeywordsDNA BINDING PROTEIN / Cas1-Cas2 integrase / CRISPR-Cas / prespacer / CRISPR leader / CRISPR repeat / spacer acquisition
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
DNA / DNA (> 10) / CRISPR-associated endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2
Similarity search - Component
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsSasnauskas, G. / Gaizauskaite, U. / Tamulaitiene, G.
Funding supportLithuania, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endoribonuclease Cas2
B: CRISPR-associated endonuclease Cas1
C: CRISPR-associated endonuclease Cas1
D: CRISPR-associated endoribonuclease Cas2
E: CRISPR-associated endonuclease Cas1
F: CRISPR-associated endonuclease Cas1
G: Prespacer DNA, chain G
H: Prespacer DNA, chain H
I: Integration target, chain I
J: Integration target, chain J
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,91012
Polymers213,86110
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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CRISPR-associated ... , 2 types, 6 molecules ADBCEF

#1: Protein CRISPR-associated endoribonuclease Cas2


Mass: 13431.560 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Gene: cas2 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: G3ECR3, Hydrolases; Acting on ester bonds
#2: Protein
CRISPR-associated endonuclease Cas1


Mass: 35363.461 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Gene: cas1 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: G3ECR2, Hydrolases; Acting on ester bonds

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Prespacer DNA, chain ... , 2 types, 2 molecules GH

#3: DNA chain Prespacer DNA, chain G


Mass: 8062.258 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Prespacer DNA, chain H


Mass: 7917.082 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Integration target, chain ... , 2 types, 2 molecules IJ

#5: DNA chain Integration target, chain I


Mass: 14729.506 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain Integration target, chain J


Mass: 14835.518 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 2 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cas1-Cas2 CRISPR integrase bound to prespacer and target DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMNaCl1
22 mMcalcium acetateCa(OAc)21
310 mMTris-HCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 29.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3129

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1+230427particle selection
2EPU3.2image acquisition
7UCSF ChimeraX1.5model fitting
8Coot0.9.8.1model fitting
13cryoSPARC4.2.1+2304273D reconstruction
14PHENIX1.21rc1_4903model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1557793 / Details: blob picking in cryoSPARC live session
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82944 / Details: Non-uniform refinement in cryoSPARC / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE

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