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Yorodumi- EMDB-27769: Type I-C Cas4-Cas1-Cas2 complex bound to a 24 bp duplex PAM/PAM p... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27769 | |||||||||
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Title | Type I-C Cas4-Cas1-Cas2 complex bound to a 24 bp duplex PAM/PAM prespacer | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR Cas adaptation type I-C / Protein-DNA complex / DNA BINDING PROTEIN | |||||||||
Biological species | Alkalihalobacillus halodurans C-125 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.9 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_27769.map.gz | 204 MB | EMDB map data format | |
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Header (meta data) | emd-27769-v30.xml emd-27769.xml | 13.4 KB 13.4 KB | Display Display | EMDB header |
Images | emd_27769.png | 75 KB | ||
Filedesc metadata | emd-27769.cif.gz | 3.8 KB | ||
Others | emd_27769_half_map_1.map.gz emd_27769_half_map_2.map.gz | 200.7 MB 200.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27769 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27769 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_27769.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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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: #1
File | emd_27769_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_27769_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/PAM substrate
Entire | Name: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate |
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Components |
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-Supramolecule #1: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate
Supramolecule | Name: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Alkalihalobacillus halodurans C-125 (bacteria) |
-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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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2 |
-Image processing
Startup model | Type of model: NONE |
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Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 160000 |