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- EMDB-36225: Cryo-EM structure of the SaCas9-sgRNA-AcrIIA15-promoter DNA dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-36225
TitleCryo-EM structure of the SaCas9-sgRNA-AcrIIA15-promoter DNA dimer
Map data
Sample
  • Complex: SaCas9-sgRNA-AcrIIA15-promoter DNA complex
    • Complex: SaCas9
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: sgRNA
      • RNA: sgRNA
    • Complex: AcrIIA15
      • Protein or peptide: AcrIIA15
    • Complex: DNA
      • DNA: DNA (25-MER)
      • DNA: DNA (25-MER)
KeywordsII-A type anti-CRISPR protein / VIRAL PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / 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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria) / Staphylococcus delphini (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.82 Å
AuthorsDeng X / Wang Y
Funding support China, 2 items
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, 2023-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36225.png

Downloads & links

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Map

FileDownload / File: emd_36225.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 360 pix.
= 374.4 Å		1.04 Å/pix.
x 360 pix.
= 374.4 Å		1.04 Å/pix.
x 360 pix.
= 374.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.17535062 - 0.60695744
Average (Standard dev.)-0.00034419532 (±0.014929133)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36225_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36225_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 : SaCas9-sgRNA-AcrIIA15-promoter DNA complex

EntireName: SaCas9-sgRNA-AcrIIA15-promoter DNA complex
Components
  • Complex: SaCas9-sgRNA-AcrIIA15-promoter DNA complex
    • Complex: SaCas9
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: sgRNA
      • RNA: sgRNA
    • Complex: AcrIIA15
      • Protein or peptide: AcrIIA15
    • Complex: DNA
      • DNA: DNA (25-MER)
      • DNA: DNA (25-MER)

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Supramolecule #1: SaCas9-sgRNA-AcrIIA15-promoter DNA complex

SupramoleculeName: SaCas9-sgRNA-AcrIIA15-promoter DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Staphylococcus aureus (bacteria)

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Supramolecule #2: SaCas9

SupramoleculeName: SaCas9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: sgRNA

SupramoleculeName: sgRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Staphylococcus delphini (bacteria)

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Supramolecule #4: AcrIIA15

SupramoleculeName: AcrIIA15 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Supramolecule #5: DNA

SupramoleculeName: DNA / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4-#5

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 124.158727 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRNYILGLD IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH SELSGINPY EARVKGLSQK LSEEEFSAAL LHLAKRRGVH NVNEVEEDTG NELSTKEQIS RNSKALEEKY VAELQLERLK K DGEVRGSI ...String:
MKRNYILGLD IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH SELSGINPY EARVKGLSQK LSEEEFSAAL LHLAKRRGVH NVNEVEEDTG NELSTKEQIS RNSKALEEKY VAELQLERLK K DGEVRGSI NRFKTSDYVK EAKQLLKVQK AYHQLDQSFI DTYIDLLETR RTYYEGPGEG SPFGWKDIKE WYEMLMGHCT YF PEELRSV KYAYNADLYN ALNDLNNLVI TRDENEKLEY YEKFQIIENV FKQKKKPTLK QIAKEILVNE EDIKGYRVTS TGK PEFTNL KVYHDIKDIT ARKEIIENAE LLDQIAKILT IYQSSEDIQE ELTNLNSELT QEEIEQISNL KGYTGTHNLS LKAI NLILD ELWHTNDNQI AIFNRLKLVP KKVDLSQQKE IPTTLVDDFI LSPVVKRSFI QSIKVINAII KKYGLPNDII IELAR EKNS KDAQKMINEM QKRNRQTNER IEEIIRTTGK ENAKYLIEKI KLHDMQEGKC LYSLEAIPLE DLLNNPFNYE VDHIIP RSV SFDNSFNNKV LVKQEENSKK GNRTPFQYLS SSDSKISYET FKKHILNLAK GKGRISKTKK EYLLEERDIN RFSVQKD FI NRNLVDTRYA TRGLMNLLRS YFRVNNLDVK VKSINGGFTS FLRRKWKFKK ERNKGYKHHA EDALIIANAD FIFKEWKK L DKAKKVMENQ MFEEKQAESM PEIETEQEYK EIFITPHQIK HIKDFKDYKY SHRVDKKPNR ELINDTLYST RKDDKGNTL IVNNLNGLYD KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKI KYYGNKLNA HLDITDDYPN SRNKVVKLSL KPYRFDVYLD NGVYKFVTVK NLDVIKKENY YEVNSKCYEE AKKLKKISNQ A EFIASFYN NDLIKINGEL YRVIGVNNDL LNRIEVNMID ITYREYLENM NDKRPPRIIK TIASKTQSIK KYSTDILGNL YE VKSKKHP QIIKKG

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #3: AcrIIA15

MacromoleculeName: AcrIIA15 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus delphini (bacteria)
Molecular weightTheoretical: 20.160963 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SMRKTIERLL NSELSSNSIA VRTGVSQAVI SKLRNGKKEL GNLTLNSAEK LFEYQKEMEK VDTWIVYRGR TADMNKSYIA EGSTYEEVY NNFVDKYGYD VLDEDIYEIQ LLKKNGENLD DYDVDSDGIN NYDKLDEFRE SDYVDLEDYD YRELFENSSS Q VYYHEFEI THE

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 31.304432 KDa
SequenceString:
GGUCUGCUAU UUCUAUUUAC GUUUUAGUAC UCUGGAAACA GAAUCUACUA AAACAAGGCA AAAUGCCGUG UUUAUCUCGU CAACUUGUU GGCGAGAUC

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Macromolecule #4: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Staphylococcus delphini (bacteria)
Molecular weightTheoretical: 7.722062 KDa
SequenceString:
(DA)(DT)(DA)(DA)(DT)(DT)(DA)(DT)(DG)(DA) (DC)(DA)(DA)(DA)(DT)(DG)(DT)(DC)(DA)(DT) (DA)(DG)(DA)(DA)(DA)

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Macromolecule #5: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Staphylococcus delphini (bacteria)
Molecular weightTheoretical: 7.627954 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DC)(DT)(DA)(DT)(DG)(DA) (DC)(DA)(DT)(DT)(DT)(DG)(DT)(DC)(DA)(DT) (DA)(DA)(DT)(DT)(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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5axw

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 72182
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

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

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