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Yorodumi- PDB-7z4l: SpCas9 bound to 18-nucleotide complementary DNA substrate in the ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7z4l | ||||||
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Title | SpCas9 bound to 18-nucleotide complementary DNA substrate in the checkpoint state | ||||||
Components |
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Keywords | HYDROLASE / CRISPR / Cas9 / R-loop / substrate binding / off-target | ||||||
Function / homology | Function and homology information maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding Similarity search - Function | ||||||
Biological species | Streptococcus pyogenes (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.54 Å | ||||||
Authors | Pacesa, M. / Jinek, M. | ||||||
Funding support | Switzerland, 1items
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Citation | Journal: Nature / Year: 2022 Title: R-loop formation and conformational activation mechanisms of Cas9. Authors: Martin Pacesa / Luuk Loeff / Irma Querques / Lena M Muckenfuss / Marta Sawicka / Martin Jinek / Abstract: Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise ...Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7z4l.cif.gz | 360.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7z4l.ent.gz | 278.4 KB | Display | PDB format |
PDBx/mmJSON format | 7z4l.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7z4l_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 7z4l_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7z4l_validation.xml.gz | 62 KB | Display | |
Data in CIF | 7z4l_validation.cif.gz | 92.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/z4/7z4l ftp://data.pdbj.org/pub/pdb/validation_reports/z4/7z4l | HTTPS FTP |
-Related structure data
Related structure data | 14501MC 7z4cC 7z4dC 7z4eC 7z4gC 7z4hC 7z4iC 7z4jC 7z4kC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA chain , 2 types, 2 molecules CD
#3: DNA chain | Mass: 11643.458 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 11734.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-RNA chain / Protein , 2 types, 2 molecules AB
#1: RNA chain | Mass: 33005.641 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#2: Protein | Mass: 158699.844 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli) References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds |
-Non-polymers , 3 types, 9 molecules
#5: Chemical | ChemComp-MG / | ||
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#6: Chemical | #7: Water | ChemComp-HOH / | |
-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide complementary DNA substrate in the inactive state Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66518 / Symmetry type: POINT | ||||||||||||||||||||||||
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