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Open data
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Basic information
Entry | Database: PDB / ID: 8d3l | |||||||||
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Title | Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/PAM prespacer | |||||||||
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![]() | HYDROLASE/DNA / CRISPR Cas adaptation type I-C / HYDROLASE-DNA complex | |||||||||
Function / homology | ![]() 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / iron-sulfur cluster binding / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / magnesium ion binding / protein homodimerization activity ...5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / iron-sulfur cluster binding / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / magnesium ion binding / protein homodimerization activity / DNA binding / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å | |||||||||
![]() | Dhingra, Y. / Suresh, S.K. / Juneja, P. / Sashital, D.G. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation. Authors: Yukti Dhingra / Shravanti K Suresh / Puneet Juneja / Dipali G Sashital / ![]() Abstract: Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of ...Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 385.5 KB | Display | ![]() |
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PDB format | ![]() | 314.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 27159MC ![]() 8d3mC ![]() 8d3pC ![]() 8d3qC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-CRISPR-associated endonuclease ... , 2 types, 6 molecules ABCDEF
#1: Protein | Mass: 39393.340 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125 Gene: cas1, BH0341 / Production host: ![]() ![]() References: UniProt: Q9KFX9, Hydrolases; Acting on ester bonds #2: Protein | Mass: 11038.668 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125 Gene: cas2, BH0342 / Production host: ![]() ![]() References: UniProt: Q9KFX8, Hydrolases; Acting on ester bonds |
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-PAM/PAM strand ... , 2 types, 2 molecules GH
#3: DNA chain | Mass: 10139.498 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 9840.351 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein , 1 types, 2 molecules IJ
#5: Protein | Mass: 25403.396 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: cas4, E2L07_14035 / Production host: ![]() ![]() References: UniProt: A0A4Y7WTW2, 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) |
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-Non-polymers , 3 types, 8 molecules ![](data/chem/img/SF4.gif)
![](data/chem/img/MN.gif)
![](data/chem/img/HOH.gif)
![](data/chem/img/MN.gif)
![](data/chem/img/HOH.gif)
#6: Chemical | #7: Chemical | #8: Water | ChemComp-HOH / | |
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-Details
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: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 Details: 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 2 mM DTT, and 2 mM MnCl2 |
Specimen | Conc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % |
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Electron microscopy imaging
Microscopy | Model: TFS GLACIOS |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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EM software | Name: cryoSPARC / Category: 3D reconstruction | ||||||||||||||||||||||||
CTF correction | Type: NONE | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124000 / Symmetry type: POINT | ||||||||||||||||||||||||
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