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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
  • sgRNA
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 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / 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 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: Nat Commun / Year: 2024
Title: An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding.
Authors: Xieshuting Deng / Wei Sun / Xueyan Li / Jiuyu Wang / Zhi Cheng / Gang Sheng / Yanli Wang /
Abstract: AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two ...AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two activities remains unclear. Here, we determined the cryo-EM structure of AcrIIA15 in complex with SaCas9-sgRNA to reveal the inhibitory mechanism of the Acr's C-terminal domain (CTD) in mimicking dsDNA to block protospacer adjacent motif (PAM) recognition. For the N-terminal domain (NTD), our crystal structures of the AcrIIA15-promoter DNA show that AcrIIA15 dimerizes through its NTD to recognize double-stranded (ds) DNA. Further, AcrIIA15 can simultaneously bind to both SaCas9-sgRNA and promoter DNA, creating a supercomplex of two Cas9s bound to two CTDs converging on a dimer of the NTD bound to a dsDNA. These findings shed light on AcrIIA15's inhibitory mechanisms and its autoregulation of transcription, enhancing our understanding of phage-host interactions and CRISPR defense.
History
DepositionMay 20, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
Water00
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


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
3sgRNACOMPLEX#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 FIELD / 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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