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- PDB-8pj9: Cas9 bound to cognate DNA, Streptococcus thermophilus DGCC 7710 C... -

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Basic information

Entry
Database: PDB / ID: 8pj9
TitleCas9 bound to cognate DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Components
  • (DNA oligoduplex, ...) x 2
  • CRISPR-associated endonuclease Cas9
  • crRNA, chain B
  • tracrRNA, chain C
KeywordsRNA BINDING PROTEIN / Cas9 / CRISPR-Cas / PAM / spacer acquisition
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.24 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
DepositionJun 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 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 endonuclease Cas9
B: crRNA, chain B
C: tracrRNA, chain C
D: DNA oligoduplex, target strand, chains D,E
E: DNA oligoduplex, target strand, chains D,E
F: DNA oligoduplex, non-target strand, chain F
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,3107
Polymers223,2866
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 2 types, 2 molecules BC

#2: RNA chain crRNA, chain B


Mass: 13609.083 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Streptococcus thermophilus DGCC 7710 (bacteria)
#3: RNA chain tracrRNA, chain C


Mass: 24119.357 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Streptococcus thermophilus DGCC 7710 (bacteria)
References: GenBank: 343794779

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DNA oligoduplex, ... , 2 types, 3 molecules DEF

#4: DNA chain DNA oligoduplex, target strand, chains D,E


Mass: 9670.199 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA oligoduplex, non-target strand, chain F


Mass: 3718.427 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Non-polymers , 2 types, 2 molecules A

#1: Protein CRISPR-associated endonuclease Cas9 / St-Cas9


Mass: 162498.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Gene: cas9, csn1
Production host: Escherichia coli str. K-12 substr. DH10B (bacteria)
References: UniProt: G3ECR1, Hydrolases; Acting on ester bonds
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / 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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplexCOMPLEX#1-#50MULTIPLE SOURCES
2CRISPR-associated endonuclease Cas9COMPLEX#11RECOMBINANT
3crRNA, chain B and tracrRNA, chain CCOMPLEX#2-#31RECOMBINANT
4DNA oligoduplex, target strand, chains D,E and DNA oligoduplex, non-target strand, chain FCOMPLEX#4-#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Streptococcus thermophilus DGCC 7710 (bacteria)1268061
33Streptococcus thermophilus DGCC 7710 (bacteria)1268061
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli str. K-12 substr. DH10B (bacteria)316385
33synthetic construct (others)32630
44synthetic construct (others)32630
Buffer solutionpH: 7.5
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
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1744

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.0.3particle selection
2EPU3image acquisition
4cryoSPARC4.0.3CTF correction
7UCSF ChimeraX1.3model fitting
8Coot0.9.8.1model fitting
13cryoSPARC4.1.13D reconstruction
14PHENIX1.21rc1_4903model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 894269 / Details: Blob picking in cryoSPARC live session
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119790 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE

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