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Yorodumi- EMDB-27161: Type I-C Cas4-Cas1-Cas2 complex bound to half-site integration in... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27161 | |||||||||
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Title | Type I-C Cas4-Cas1-Cas2 complex bound to half-site integration intermediate (HSI) | |||||||||
Map data | ||||||||||
Sample |
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Function / homology | Function and homology information 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 | Alkalihalobacillus halodurans C-125 (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.26 Å | |||||||||
Authors | Dhingra Y / Suresh SK / Juneja P / Sashital DG | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Mol Cell / Year: 2022 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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27161.map.gz | 135.3 MB | EMDB map data format | |
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Header (meta data) | emd-27161-v30.xml emd-27161.xml | 21.5 KB 21.5 KB | Display Display | EMDB header |
Images | emd_27161.png | 50 KB | ||
Others | emd_27161_half_map_1.map.gz emd_27161_half_map_2.map.gz | 200.7 MB 200.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27161 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27161 | HTTPS FTP |
-Validation report
Summary document | emd_27161_validation.pdf.gz | 1 MB | Display | EMDB validaton report |
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Full document | emd_27161_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | emd_27161_validation.xml.gz | 15.6 KB | Display | |
Data in CIF | emd_27161_validation.cif.gz | 17.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27161 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27161 | HTTPS FTP |
-Related structure data
Related structure data | 8d3pMC 8d3lC 8d3mC 8d3qC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_27161.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.9063 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_27161_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27161_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
+Supramolecule #1: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
+Macromolecule #1: CRISPR-associated endonuclease Cas1
+Macromolecule #2: CRISPR-associated endonuclease Cas2
+Macromolecule #5: CRISPR-associated exonuclease Cas4
+Macromolecule #3: HSI strand 2- integrated prespacer strand plus repeat
+Macromolecule #4: HSI strand 2
+Macromolecule #6: HSI strand 3 bottom strand leader-repeat
+Macromolecule #7: HSI strand 4 top leader strand
+Macromolecule #8: IRON/SULFUR CLUSTER
+Macromolecule #9: MANGANESE (II) ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.75 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 2 mM DTT, and 2 mM MnCl2 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
-Image processing
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 4.26 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 182000 |
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Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |