+Open data
-Basic information
Entry | Database: PDB / ID: 7z4k | ||||||
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Title | SpCas9 bound to 10-nucleotide complementary DNA substrate | ||||||
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: 3.81 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 | 7z4k.cif.gz | 323.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7z4k.ent.gz | 251 KB | Display | PDB format |
PDBx/mmJSON format | 7z4k.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7z4k_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 7z4k_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 7z4k_validation.xml.gz | 57.8 KB | Display | |
Data in CIF | 7z4k_validation.cif.gz | 85.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/z4/7z4k ftp://data.pdbj.org/pub/pdb/validation_reports/z4/7z4k | HTTPS FTP |
-Related structure data
Related structure data | 14500MC 7z4cC 7z4dC 7z4eC 7z4gC 7z4hC 7z4iC 7z4jC 7z4lC 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
#1: Protein | Mass: 158588.781 Da / Num. of mol.: 1 / Mutation: D10A, H840A 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 |
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#2: RNA chain | Mass: 33005.641 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: DNA chain | Mass: 9786.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#4: DNA chain | Mass: 9822.324 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-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 10-nucleotide complementary DNA substrate Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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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: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Image recording | Electron dose: 66.22 e/Å2 / Film or detector model: GATAN K3 (6k 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: 3.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127523 / Symmetry type: POINT |