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- PDB-8jg9: Cryo-EM structure of the SaCas9-sgRNA-AcrIIA15-promoter DNA dimer -

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Basic information

Entry
Database: PDB / ID: 8jg9
TitleCryo-EM structure of the SaCas9-sgRNA-AcrIIA15-promoter DNA dimer
Components
  • (DNA (25-MER)) x 2
  • AcrIIA15
  • CRISPR-associated endonuclease Cas9
  • sgRNASubgenomic mRNA
KeywordsVIRAL PROTEIN / II-A type anti-CRISPR protein
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, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / 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 speciesStaphylococcus aureus (bacteria)
Staphylococcus delphini (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.82 Å
AuthorsDeng, X. / Wang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31930065 China
National Natural Science Foundation of China (NSFC)31725008 China
CitationJournal: To Be Published
Title: Cryo-EM structure of the SaCas9-sgRNA-AcrIIA15-promoter DNA dimer
Authors: Deng, X. / Wang, Y.
History
DepositionMay 20, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 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: sgRNA
D: CRISPR-associated endonuclease Cas9
E: sgRNA
F: AcrIIA15
G: DNA (25-MER)
H: DNA (25-MER)
C: AcrIIA15


Theoretical massNumber of molelcules
Total (without water)366,5988
Polymers366,5988
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9 / SaCas9


Mass: 124158.727 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: cas9 / Production host: Escherichia coli (E. coli)
References: UniProt: J7RUA5, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA / Subgenomic mRNA


Mass: 31304.432 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli DH5[alpha] (bacteria)
#3: Protein AcrIIA15


Mass: 20160.963 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus delphini (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: DNA chain DNA (25-MER)


Mass: 7722.062 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus delphini (bacteria)
#5: DNA chain DNA (25-MER)


Mass: 7627.954 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus delphini (bacteria)

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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
1SaCas9-sgRNA-AcrIIA15-promoter DNA complexCOMPLEXall0MULTIPLE SOURCES
2SaCas9COMPLEX#11RECOMBINANT
3sgRNASubgenomic mRNACOMPLEX#21RECOMBINANT
4AcrIIA15COMPLEX#31RECOMBINANT
5DNACOMPLEX#4-#51SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Staphylococcus aureus (bacteria)1280
33Staphylococcus delphini (bacteria)53344
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli DH5[alpha] (bacteria)6683
44Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72182 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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