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Open data
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Basic information
Entry | Database: PDB / ID: 7mi4 | ||||||
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Title | Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex | ||||||
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![]() | HYDROLASE/DNA / CRISPR/Cas / Cas4 / PAM recognition / HYDROLASE-DNA complex | ||||||
Function / homology | ![]() 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 | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
![]() | 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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PDBx/mmCIF format | ![]() | 379.8 KB | Display | ![]() |
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PDB format | ![]() | 317.5 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 919.3 KB | Display | ![]() |
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Full document | ![]() | 964.3 KB | Display | |
Data in XML | ![]() | 60 KB | Display | |
Data in CIF | ![]() | 89.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 23839MC ![]() 7mi5C ![]() 7mi9C ![]() 7mibC ![]() 7midC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 62598.496 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() 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.) ![]() ![]() ![]() References: UniProt: Q74H35, Hydrolases; Acting on ester bonds #3: DNA chain | Mass: 10769.917 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) ![]() #4: Chemical | #5: Chemical | ChemComp-MN / Has ligand of interest | Y | |
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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 |
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Sample preparation
Component | Name: Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex Type: COMPLEX / Details: Cas4 recognizes PAM / Entity ID: #1-#3 / 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 / Formula: NaCl |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: normal / 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 / 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 |
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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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120000 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: OTHER | ||||||||||||||||||||||||
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