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Yorodumi- PDB-8fy9: Cryo-EM structure of Cas1:Cas2-DEDDh:PAM-deficient prespacer complex -
+Open data
-Basic information
Entry | Database: PDB / ID: 8fy9 | ||||||||||||
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Title | Cryo-EM structure of Cas1:Cas2-DEDDh:PAM-deficient prespacer complex | ||||||||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / CRISPR / integrase / CRISPR adaptation module / PAM / prespacer / exonuclease / DNA binding protein-DNA complex / enzyme / ribonucleoprotein | ||||||||||||
Function / homology | DNA / DNA (> 10) Function and homology information | ||||||||||||
Biological species | Megasphaera (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||||||||
Authors | Skopintsev, P. / Tuck, O.T. / Soczek, K.M. / Doudna, J. | ||||||||||||
Funding support | United States, Switzerland, 3items
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Citation | Journal: Nature / Year: 2023 Title: Genome expansion by a CRISPR trimmer-integrase. Authors: Joy Y Wang / Owen T Tuck / Petr Skopintsev / Katarzyna M Soczek / Gary Li / Basem Al-Shayeb / Julia Zhou / Jennifer A Doudna / Abstract: CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems ...CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8fy9.cif.gz | 258.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8fy9.ent.gz | 200.4 KB | Display | PDB format |
PDBx/mmJSON format | 8fy9.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8fy9_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8fy9_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8fy9_validation.xml.gz | 48.6 KB | Display | |
Data in CIF | 8fy9_validation.cif.gz | 72 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/fy/8fy9 ftp://data.pdbj.org/pub/pdb/validation_reports/fy/8fy9 | HTTPS FTP |
-Related structure data
Related structure data | 29561MC 8fyaC 8fybC 8fycC 8fydC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 32632.615 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Megasphaera (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta #2: Protein | Mass: 35022.074 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Megasphaera (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta #3: DNA chain | | Mass: 8629.578 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Megasphaera (bacteria) #4: DNA chain | | Mass: 8661.576 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Megasphaera (bacteria) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cas1:Cas2-DEDDh:PAM-deficient prespacer complex / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.222 MDa / Experimental value: NO |
Source (natural) | Organism: Megasphaera (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 461266 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
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