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Yorodumi- EMDB-23843: Full integration complex of Cas1/Cas2 from Cas4-containing system -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23843 | |||||||||
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Title | Full integration complex of Cas1/Cas2 from Cas4-containing system | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR/Cas / Cas4 / PAM recognition / full integration / HYDROLASE-DNA complex | |||||||||
Function / homology | Function and homology information 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 | Geobacter sulfurreducens (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.89 Å | |||||||||
Authors | Hu CY / Ke AK | |||||||||
Citation | Journal: Nature / Year: 2021 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. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23843.map.gz | 64.3 MB | EMDB map data format | |
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Header (meta data) | emd-23843-v30.xml emd-23843.xml | 23.6 KB 23.6 KB | Display Display | EMDB header |
Images | emd_23843.png | 111.9 KB | ||
Filedesc metadata | emd-23843.cif.gz | 6.5 KB | ||
Others | emd_23843_additional_1.map.gz emd_23843_additional_2.map.gz emd_23843_additional_3.map.gz | 63.3 MB 63.4 MB 54.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23843 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23843 | HTTPS FTP |
-Validation report
Summary document | emd_23843_validation.pdf.gz | 528.4 KB | Display | EMDB validaton report |
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Full document | emd_23843_full_validation.pdf.gz | 528 KB | Display | |
Data in XML | emd_23843_validation.xml.gz | 6 KB | Display | |
Data in CIF | emd_23843_validation.cif.gz | 6.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23843 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23843 | HTTPS FTP |
-Related structure data
Related structure data | 7mi9MC 7mi4C 7mi5C 7mibC 7midC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_23843.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.314 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: #1
File | emd_23843_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: #2
File | emd_23843_additional_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: #3
File | emd_23843_additional_3.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Full integration complex of Cas1/Cas2 from Cas4-containing system
+Supramolecule #1: Full integration complex of Cas1/Cas2 from Cas4-containing system
+Supramolecule #2: Local refinement half map (spacer side)
+Supramolecule #3: Local refinement half map (leader side)
+Supramolecule #4: low resolution map before local refinement
+Macromolecule #1: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
+Macromolecule #2: CRISPR-associated endoribonuclease Cas2
+Macromolecule #3: DNA (80-MER)
+Macromolecule #4: DNA (72-MER)
+Macromolecule #5: DNA (5'-D(P*CP*GP*GP*AP*AP*AP*AP*GP*AP*GP*CP*C)-3')
+Macromolecule #6: DNA (5'-D(P*GP*AP*AP*GP*CP*C)-3')
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.0 mg/mL |
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Buffer | pH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride / Details: WITH 5 mM DTT |
Grid | Model: Quantifoil R1.2/1.3 / Support film - Material: CARBON |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 6 seconds. |
-Electron microscopy
Microscope | TFS TALOS |
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Specialist optics | Phase plate: VOLTA PHASE PLATE |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1200 / Average exposure time: 0.35 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus min: 1.5 µm |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: OTHER |
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Output model | PDB-7mi9: |