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Yorodumi- EMDB-23849: Half integration complex of Cas1-Cas2 in Cas4 containing system -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23849 | |||||||||
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Title | Half integration complex of Cas1-Cas2 in Cas4 containing system | |||||||||
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 / 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: 5.8 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_23849.map.gz | 34.3 MB | EMDB map data format | |
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Header (meta data) | emd-23849-v30.xml emd-23849.xml | 12.5 KB 12.5 KB | Display Display | EMDB header |
Images | emd_23849.png | 98.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23849 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23849 | HTTPS FTP |
-Related 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_23849.map.gz / Format: CCP4 / Size: 36.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.32 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Half integration complex and Cas4 dissociated from PAM side
Entire | Name: Half integration complex and Cas4 dissociated from PAM side |
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Components |
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-Supramolecule #1: Half integration complex and Cas4 dissociated from PAM side
Supramolecule | Name: Half integration complex and Cas4 dissociated from PAM side type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: half integration in cas4 containing system |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA |
Molecular weight | Experimental: 300 KDa |
-Macromolecule #1: half integration complex of Cas1-Cas2
Macromolecule | Name: half integration complex of Cas1-Cas2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MAETDGSIPL IPVRMLNEHV YCPRLAYLMW VQGEFSHNEF TVDGVIRHRR VDAGGGVLPS ETQEDSRIH ARSVSLSSER LGITAKIDLV EGEGAYVSPV DYKRGKRPHV AGGAYEPERV Q LCAQGLLL REHGFASDGG ALYFVASRER VPVAFDDELI GRTLAAIDEM ...String: MAETDGSIPL IPVRMLNEHV YCPRLAYLMW VQGEFSHNEF TVDGVIRHRR VDAGGGVLPS ETQEDSRIH ARSVSLSSER LGITAKIDLV EGEGAYVSPV DYKRGKRPHV AGGAYEPERV Q LCAQGLLL REHGFASDGG ALYFVASRER VPVAFDDELI GRTLAAIDEM GRTALSGTMP PP LEDSPKC PRCSLVGICL PDEVRFLSHL SVEPRPIIPA DGRGLPLYVQ SPKAYVRKDG DCL VIEEER VRVAEARLGE TSQVALFGNA TLTTAALHEC LRREIPVTWL SYGGWFMGHT VSTG HRNVE TRTYQYQRSF DPETCLNLAR RWIVAKIANC RTLLRRNWRG EGDEAKAPPG LLMSL QDDM RHAMRAPSLE VLLGIEGASA GRYFQHFSRM LRGGDGEGMG FDFTTRNRRP PKDPVN ALL SFAYAMLTRE WTVALAAVGL DPYRGFYHQP RFGRPALALD MMEPFRPLIA DSTVLMA IN NGEIRTGDFV RSAGGCNLTD SARKRFIAGF ERRMEQEVTH PIFKYTISYR RLLEVQAR L LTRYLSGEIP AYPNFVTR |
-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 / Material: COPPER / Mesh: 300 / 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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