+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23839 | |||||||||
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Title | Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex | |||||||||
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 / defense response to virus / endonuclease activity / 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: 3.2 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_23839.map.gz | 56.4 MB | EMDB map data format | |
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Header (meta data) | emd-23839-v30.xml emd-23839.xml | 15 KB 15 KB | Display Display | EMDB header |
Images | emd_23839.png | 145.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23839 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23839 | HTTPS FTP |
-Validation report
Summary document | emd_23839_validation.pdf.gz | 451.4 KB | Display | EMDB validaton report |
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Full document | emd_23839_full_validation.pdf.gz | 451 KB | Display | |
Data in XML | emd_23839_validation.xml.gz | 5.8 KB | Display | |
Data in CIF | emd_23839_validation.cif.gz | 6.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23839 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23839 | HTTPS FTP |
-Related structure data
Related structure data | 7mi4MC 7mi5C 7mi9C 7mibC 7midC M: atomic model generated by this map C: citing same article (ref.) |
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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_23839.map.gz / Format: CCP4 / Size: 59.6 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: 1.23 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 : Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Entire | Name: Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex |
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Components |
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-Supramolecule #1: Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Supramolecule | Name: Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: Cas4 recognizes PAM |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) |
Molecular weight | Experimental: 300 KDa |
-Macromolecule #1: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
Macromolecule | Name: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA |
Molecular weight | Theoretical: 62.598496 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MAETDGSIPL IPVRMLNEHV YCPRLAYLMW VQGEFSHNEF TVDGVIRHRR VDAGGGVLPS ETQEDSRIHA RSVSLSSERL GITAKIDLV EGEGAYVSPV DYKRGKRPHV AGGAYEPERV QLCAQGLLLR EHGFASDGGA LYFVASRERV PVAFDDELIG R TLAAIDEM ...String: MAETDGSIPL IPVRMLNEHV YCPRLAYLMW VQGEFSHNEF TVDGVIRHRR VDAGGGVLPS ETQEDSRIHA RSVSLSSERL GITAKIDLV EGEGAYVSPV DYKRGKRPHV AGGAYEPERV QLCAQGLLLR EHGFASDGGA LYFVASRERV PVAFDDELIG R TLAAIDEM GRTALSGTMP PPLEDSPKCP RCSLVGICLP DEVRFLSHLS VEPRPIIPAD GRGLPLYVQS PKAYVRKDGD CL VIEEERV RVAEARLGET SQVALFGNAT LTTAALHECL RREIPVTWLS YGGWFMGHTV STGHRNVETR TYQYQRSFDP ETC LNLARR WIVAKIANCR TLLRRNWRGE GDEAKAPPGL LMSLQDDMRH AMRAPSLEVL LGIEGASAGR YFQHFSRMLR GGDG EGMGF DFTTRNRRPP KDPVNALLSF AYAMLTREWT VALAAVGLDP YRGFYHQPRF GRPALALDMM EPFRPLIADS TVLMA INNG EIRTGDFVRS AGGCNLTDSA RKRFIAGFER RMEQEVTHPI FKYTISYRRL LEVQARLLTR YLSGEIPAYP NFVTR |
-Macromolecule #2: CRISPR-associated endoribonuclease Cas2
Macromolecule | Name: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA |
Molecular weight | Theoretical: 11.190176 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MEHLYIVSYD IRNQRRWRRL FKTMHGFGCW LQLSVFQCRL DRIRIIKMEA AINEIVNHAE DHVLILDLGP AENVKPKVSS IGKTFDPIL RQAVIV |
-Macromolecule #3: DNA (35-MER)
Macromolecule | Name: DNA (35-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: Geobacter sulfurreducens (bacteria) |
Molecular weight | Theoretical: 10.769917 KDa |
Sequence | String: (DG)(DT)(DC)(DG)(DT)(DA)(DG)(DC)(DT)(DG) (DA)(DG)(DG)(DC)(DC)(DT)(DC)(DA)(DG)(DC) (DT)(DA)(DC)(DG)(DA)(DC)(DT)(DT)(DT) (DT)(DT)(DG)(DA)(DA)(DT) |
-Macromolecule #4: IRON/SULFUR CLUSTER
Macromolecule | Name: IRON/SULFUR CLUSTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: SF4 |
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Molecular weight | Theoretical: 351.64 Da |
Chemical component information | ChemComp-FS1: |
-Macromolecule #5: MANGANESE (II) ION
Macromolecule | Name: MANGANESE (II) ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: MN |
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Molecular weight | Theoretical: 54.938 Da |
-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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.38 kPa / Details: normal |
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-7mi4: |