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Yorodumi- PDB-7mi9: Full integration complex of Cas1/Cas2 from Cas4-containing system -
+Open data
-Basic information
Entry | Database: PDB / ID: 7mi9 | ||||||
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Title | Full integration complex of Cas1/Cas2 from Cas4-containing system | ||||||
Components |
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Keywords | HYDROLASE/DNA / CRISPR/Cas / Cas4 / PAM recognition / full integration / HYDROLASE-DNA complex | ||||||
Function / homology | Function and homology information 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / RNA endonuclease activity / 4 iron, 4 sulfur cluster binding / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding Similarity search - Function | ||||||
Biological species | Geobacter sulfurreducens (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.89 Å | ||||||
Authors | Hu, C.Y. / Ke, A.K. | ||||||
Citation | Journal: Nature / Year: 2021 Title: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas. Authors: Chunyi Hu / Cristóbal Almendros / Ki Hyun Nam / Ana Rita Costa / Jochem N A Vink / Anna C Haagsma / Saket R Bagde / Stan J J Brouns / Ailong Ke / Abstract: Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A ...Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7mi9.cif.gz | 365.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7mi9.ent.gz | 288.7 KB | Display | PDB format |
PDBx/mmJSON format | 7mi9.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7mi9_validation.pdf.gz | 961.8 KB | Display | wwPDB validaton report |
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Full document | 7mi9_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 7mi9_validation.xml.gz | 61.1 KB | Display | |
Data in CIF | 7mi9_validation.cif.gz | 91.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/mi/7mi9 ftp://data.pdbj.org/pub/pdb/validation_reports/mi/7mi9 | HTTPS FTP |
-Related structure data
Related structure data | 23843MC 7mi4C 7mi5C 7mibC 7midC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-CRISPR-associated ... , 2 types, 6 molecules ABCDEF
#1: Protein | Mass: 62598.496 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA / Gene: cas4-cas1, GSU0057 / Production host: Escherichia coli (E. coli) References: UniProt: Q74H36, Hydrolases; Acting on ester bonds, 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) #2: Protein | Mass: 11190.176 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA / Gene: cas2, GSU0058 / Production host: Escherichia coli (E. coli) References: UniProt: Q74H35, Hydrolases; Acting on ester bonds |
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-DNA chain , 4 types, 4 molecules GHIJ
#3: DNA chain | Mass: 24664.730 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria) |
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#4: DNA chain | Mass: 22123.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria) |
#5: DNA chain | Mass: 3705.445 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria) |
#6: DNA chain | Mass: 1818.231 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 / Details: WITH 5 mM DTT | |||||||||||||||||||||||||||||||||||
Buffer component | Conc.: 150 mM / Name: sodium chloride / Formula: NaCl | |||||||||||||||||||||||||||||||||||
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 6 seconds |
-Electron microscopy imaging
Microscopy | Model: TFS TALOS |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: DIFFRACTION / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Image recording | Average exposure time: 0.35 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1200 |
EM imaging optics | Phase plate: VOLTA PHASE PLATE |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80000 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: OTHER | ||||||||||||||||||||||||
Refine LS restraints |
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