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Yorodumi- PDB-7pla: Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7pla | |||||||||
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Title | Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA target | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN / Cas12k / sgRNA / target DNA / Protein-RNA-DNA complex / Tn7 / Transposition / CRISPR | |||||||||
Function / homology | DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / ShCas12k Function and homology information | |||||||||
Biological species | Scytonema hofmannii (bacteria) synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å | |||||||||
Authors | Schmitz, M. / Jinek, M. | |||||||||
Funding support | European Union, 2items
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Citation | Journal: Nature / Year: 2021 Title: Target site selection and remodelling by type V CRISPR-transposon systems. Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek / Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7pla.cif.gz | 209.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7pla.ent.gz | 153.9 KB | Display | PDB format |
PDBx/mmJSON format | 7pla.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pl/7pla ftp://data.pdbj.org/pub/pdb/validation_reports/pl/7pla | HTTPS FTP |
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-Related structure data
Related structure data | 13486MC 7oxdC 7plhC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 73421.773 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8X6EH11 |
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#2: RNA chain | Mass: 82376.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria) |
#3: DNA chain | Mass: 15301.837 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#4: DNA chain | Mass: 15497.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-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: Ternary complex of ShCas12k with sgRNA and target dsDNA Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.187 MDa / Experimental value: NO |
Source (natural) | Organism: Scytonema hofmannii (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 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 51.81 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138000 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 185.47 Å2 | ||||||||||||||||||||||||
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