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Yorodumi- PDB-7lyt: Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phospho... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7lyt | ||||||
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Title | Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phosphorothioate-DNA | ||||||
Components |
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Keywords | VIRAL PROTEIN/RNA/DNA / CRISPR / CasPhi / Cas12j / Nuclease / R-loop / crRNA / PAM / RNP / Complex / VIRAL PROTEIN-RNA-DNA / VIRAL PROTEIN-RNA-DNA complex | ||||||
Function / homology | DNA / DNA (> 10) / RNA / RNA (> 10) Function and homology information | ||||||
Biological species | Biggievirus Mos11 | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
Authors | Pausch, P. / Soczek, K. / Nogales, E. / Doudna, J. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Struct Mol Biol / Year: 2021 Title: DNA interference states of the hypercompact CRISPR-CasΦ effector. Authors: Patrick Pausch / Katarzyna M Soczek / Dominik A Herbst / Connor A Tsuchida / Basem Al-Shayeb / Jillian F Banfield / Eva Nogales / Jennifer A Doudna / Abstract: CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and ...CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing. | ||||||
History |
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-Structure visualization
Movie |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7lyt.cif.gz | 322.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7lyt.ent.gz | 252.6 KB | Display | PDB format |
PDBx/mmJSON format | 7lyt.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7lyt_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 7lyt_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7lyt_validation.xml.gz | 36.3 KB | Display | |
Data in CIF | 7lyt_validation.cif.gz | 53.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ly/7lyt ftp://data.pdbj.org/pub/pdb/validation_reports/ly/7lyt | HTTPS FTP |
-Related structure data
Related structure data | 23601MC 7lysC 7m5oC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA chain , 2 types, 2 molecules CD
#3: DNA chain | Mass: 13615.714 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Biggievirus Mos11 |
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#4: DNA chain | Mass: 13072.435 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Biggievirus Mos11 |
-Protein / RNA chain , 2 types, 2 molecules AB
#1: Protein | Mass: 86127.188 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Biggievirus Mos11 / Plasmid: pPP085 / Details (production host): Addgene #158795 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star |
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#2: RNA chain | Mass: 14481.651 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Biggievirus Mos11 |
-Non-polymers , 2 types, 3 molecules
#5: Chemical | ChemComp-ZN / |
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#6: Chemical |
-Details
Has ligand of interest | Y |
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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: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in the presence of the nuclease magnesium cofactor Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.128 MDa / Experimental value: NO |
Source (natural) | Organism: Biggievirus Mos11 |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / 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 |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
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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: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 410553 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 67.58 Å2 | ||||||||||||||||||||||||
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