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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-23847 | |||||||||
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Title | Sub-complex of Cas4-Cas1-Cas2 bound PAM containing DNA | |||||||||
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Function / homology | ![]() 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 | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.56 Å | |||||||||
![]() | Hu CY / Ke AK | |||||||||
![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 16.5 KB 16.5 KB | Display Display | ![]() |
Images | ![]() | 115.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7midMC ![]() 7mi4C ![]() 7mi5C ![]() 7mi9C ![]() 7mibC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.42578 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Sub-complex of Cas4-Cas1-Cas2 bound PAM-containing DNA
Entire | Name: Sub-complex of Cas4-Cas1-Cas2 bound PAM-containing DNA |
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Components |
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-Supramolecule #1: Sub-complex of Cas4-Cas1-Cas2 bound PAM-containing DNA
Supramolecule | Name: Sub-complex of Cas4-Cas1-Cas2 bound PAM-containing DNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: Cas4-Cas1-Cas2 searching PAM-containing DNA |
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Source (natural) | Organism: ![]() |
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: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 62.598496 KDa |
Recombinant expression | Organism: ![]() ![]() |
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: ![]() |
Molecular weight | Theoretical: 11.190176 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MEHLYIVSYD IRNQRRWRRL FKTMHGFGCW LQLSVFQCRL DRIRIIKMEA AINEIVNHAE DHVLILDLGP AENVKPKVSS IGKTFDPIL RQAVIV |
-Macromolecule #3: DNA (5'-D(P*CP*AP*CP*CP*AP*TP*CP*GP*TP*GP*AP*GP*GP*CP*CP*TP*CP*AP...
Macromolecule | Name: DNA (5'-D(P*CP*AP*CP*CP*AP*TP*CP*GP*TP*GP*AP*GP*GP*CP*CP*TP*CP*AP*GP*CP*TP*AP*CP*G)-3') type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 11.050084 KDa |
Sequence | String: (DC)(DA)(DC)(DC)(DA)(DT)(DC)(DG)(DT)(DG) (DA)(DG)(DG)(DC)(DC)(DT)(DC)(DA)(DG)(DC) (DT)(DA)(DC)(DG)(DA)(DT)(DT)(DT)(DT) (DT)(DG)(DG)(DG)(DT)(DT)(DT) |
-Macromolecule #4: DNA (33-MER)
Macromolecule | Name: DNA (33-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 11.418327 KDa |
Sequence | String: (DG)(DT)(DC)(DG)(DT)(DA)(DG)(DC)(DT)(DG) (DA)(DG)(DG)(DC)(DC)(DT)(DC)(DA)(DC)(DG) (DA)(DT)(DG)(DG)(DA)(DC)(DT)(DT)(DT) (DT)(DT)(DG)(DA)(DA)(DT)(DT)(DT) |
-Macromolecule #5: IRON/SULFUR CLUSTER
Macromolecule | Name: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4 |
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Molecular weight | Theoretical: 351.64 Da |
Chemical component information | ![]() ChemComp-FS1: |
-Macromolecule #6: MANGANESE (II) ION
Macromolecule | Name: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MN |
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Molecular weight | Theoretical: 54.938 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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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. |
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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: ![]() |
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 |
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Image processing
-Atomic model buiding 1
Refinement | Protocol: OTHER |
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Output model | ![]() PDB-7mid: |