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Open data
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Basic information
Entry | Database: PDB / ID: 7mi5 | ||||||
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Title | Asymmetrical PAM-Non PAM prespacer bound Cas4/Cas1/Cas2 complex | ||||||
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![]() | HYDROLASE/DNA / ![]() ![]() | ||||||
Function / homology | ![]() 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | Hu, C.Y. / Ke, A.K. | ||||||
![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 349.8 KB | Display | ![]() |
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PDB format | ![]() | 290 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 23840MC ![]() 7mi4C ![]() 7mi9C ![]() 7mibC ![]() 7midC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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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.) ![]() ![]() ![]() ![]() ![]() References: UniProt: Q74H36, ![]() #2: Protein | Mass: 11190.176 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() References: UniProt: Q74H35, ![]() |
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-DNA chain , 2 types, 2 molecules GH
#3: DNA chain | Mass: 11339.267 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#4: DNA chain | Mass: 11418.327 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-Non-polymers , 2 types, 7 molecules ![](data/chem/img/SF4.gif)
![](data/chem/img/MN.gif)
![](data/chem/img/MN.gif)
#5: Chemical | ChemComp-SF4 / ![]() |
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#6: Chemical | ChemComp-MN / |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex Type: COMPLEX / Details: Cas4 recognizes PAM / Entity ID: #1, #3-#4 / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.3 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 7.5 / Details: with 5 mM DTT |
Buffer component | Conc.: 150 mM / Name: sodium chloride![]() ![]() |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / 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 |
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Electron microscopy imaging
Microscopy | Model: TFS TALOS |
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Electron gun | Electron source![]() ![]() |
Electron lens | Mode: DIFFRACTION![]() ![]() |
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 |
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Processing
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80000 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: OTHER |