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- EMDB-20130: Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex in presen... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-20130 | |||||||||
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Title | Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex in presence of prespacer DNA | |||||||||
![]() | Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex with prespacer DNA | |||||||||
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Biological species | ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 24.4 Å | |||||||||
![]() | Sashital DG / Lee H / Dhingra Y | |||||||||
![]() | ![]() Title: The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation. Authors: Hayun Lee / Yukti Dhingra / Dipali G Sashital / ![]() Abstract: CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional ...CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4. | |||||||||
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 | ![]() | 1.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 16.6 KB 16.6 KB | Display Display | ![]() |
Images | ![]() | 43.5 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 78.1 KB | Display | ![]() |
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Full document | ![]() | 77.2 KB | Display | |
Data in XML | ![]() | 494 B | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex with prespacer DNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.8 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 : Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex with pres...
Entire | Name: Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex with prespacer DNA |
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Components |
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-Supramolecule #1: Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex with pres...
Supramolecule | Name: Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex with prespacer DNA type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 260 KDa |
-Macromolecule #1: Cas1
Macromolecule | Name: Cas1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITF LTKNGRFLAR VVGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS K WMLERMTR EHPLRVNVEQ FKATSQLLSV MMQEIRNCDS LESLRGWEGQ ...String: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITF LTKNGRFLAR VVGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS K WMLERMTR EHPLRVNVEQ FKATSQLLSV MMQEIRNCDS LESLRGWEGQ AAINYNKVFD QM ILQQKEE FAFHGRSRRP PKDNVNAMLS FAYTLLANDV AAALETVGLD AYVGFMHQDR PGR ASLALD LMEELRGLYA DRFVLSLINR KEMTADGFYK KENGAVLMTD EARKTFLKAW QTKK QEKIT HPYLGEKMSW GLVPYVQALL LARFLRGDLD EYPPFLWK |
-Macromolecule #2: Cas2
Macromolecule | Name: Cas2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MLVLITYDVQ TSSMGGTKRL RKVAKACQNY GQRVQNSVFE CIVDSTQLTS LKLELTSLID EEKDSLRIY RLGNNYKTKV EHIGAKPSID LEDPLIF |
-Macromolecule #3: Cas4
Macromolecule | Name: Cas4 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MASNEEDRYL MLSGLQHFQF CKRQWALIHI EQQWEENVRT IEGQHLHKKA DQPFMKEKRG SKLTVRAMPI QSKNLQISGI CDVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA LRDKVVQMAK ...String: MASNEEDRYL MLSGLQHFQF CKRQWALIHI EQQWEENVRT IEGQHLHKKA DQPFMKEKRG SKLTVRAMPI QSKNLQISGI CDVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA LRDKVVQMAK EMHHYYENRH TPKVKTGPFC NNCSLQSICL PKLMNKRSVK RYIEGRLSE |
-Macromolecule #4: prespacer top strand
Macromolecule | Name: prespacer top strand / type: dna / ID: 4 / Classification: DNA |
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Source (natural) | Organism: unidentified (others) |
Sequence | String: CGTAGCTGAG GACCACCAGA ACAGTTTTGA ATTTTTTTT |
-Macromolecule #5: prespacer bottom strand
Macromolecule | Name: prespacer bottom strand / type: dna / ID: 5 / Classification: DNA |
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Source (natural) | Organism: unidentified (others) |
Sequence | String: CTGTTCTGGT GGTCCTCAGC TACGTTTTGA ATTTTTTTT |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.026 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.5 Component:
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Staining | Type: NEGATIVE / Material: uranyl formate Details: 3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed ...Details: 3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed by immediate application of 3 uL 2% (w/v) uranyl formate. The excess stain was blotted, followed by immediate application of 3 uL 2% uranyl formate. This step was repeated once more. The grids were allowed to dry for at least 5 minutes prior to imaging. | ||||||||||||||||||
Grid | Support film - Material: CARBON / Support film - topology: CONTINUOUS / Details: unspecified |
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Electron microscopy
Microscope | JEOL 2100 |
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Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 93 / Average exposure time: 0.5 sec. / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |