+Open data
-Basic information
Entry | Database: PDB / ID: 8bd5 | |||||||||||||||
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Title | Cas12k-sgRNA-dsDNA-S15-TniQ-TnsC transposon recruitment complex | |||||||||||||||
Components |
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Keywords | RNA BINDING PROTEIN / Cas12k / sgRNA / S15 / TniQ / TnsC / CRISPR-Cas / Tn7-like transposons / transposition | |||||||||||||||
Function / homology | Function and homology information small ribosomal subunit rRNA binding / regulation of translation / ribosomal small subunit assembly / cytosolic small ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / translation / cytosol / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Scytonema hofmannii (bacteria) Escherichia coli K-12 (bacteria) synthetic construct (others) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||||||||
Authors | Schmitz, M. / Querques, I. / Oberli, S. / Chanez, C. / Jinek, M. | |||||||||||||||
Funding support | Switzerland, European Union, United States, 4items
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Citation | Journal: Cell / Year: 2022 Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex. Authors: Michael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek / Abstract: CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads. | |||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8bd5.cif.gz | 635.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8bd5.ent.gz | 507.6 KB | Display | PDB format |
PDBx/mmJSON format | 8bd5.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8bd5_validation.pdf.gz | 1.9 MB | Display | wwPDB validaton report |
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Full document | 8bd5_full_validation.pdf.gz | 2 MB | Display | |
Data in XML | 8bd5_validation.xml.gz | 97.2 KB | Display | |
Data in CIF | 8bd5_validation.cif.gz | 145.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bd/8bd5 ftp://data.pdbj.org/pub/pdb/validation_reports/bd/8bd5 | HTTPS FTP |
-Related structure data
Related structure data | 15975MC 8bd4C 8bd6C 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
-Protein , 4 types, 10 molecules AEFGHIJKQX
#1: Protein | Mass: 79156.773 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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#5: Protein | Mass: 31444.617 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL3 #6: Protein | | Mass: 19011.240 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: A0A8J0PCL5 #7: Protein | | Mass: 10290.816 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpsO, secC, b3165, JW3134 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ADZ4 |
-RNA chain , 1 types, 1 molecules B
#2: RNA chain | Mass: 82376.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria) |
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-DNA chain , 2 types, 2 molecules CD
#3: DNA chain | Mass: 14916.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 15125.739 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 3 types, 16 molecules
#8: Chemical | ChemComp-ATP / #9: Chemical | ChemComp-MG / #10: Chemical | |
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-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: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitment complex Type: COMPLEX / Details: Cas12k, sgRNA, target DNA, S15, TniQ, TnsC / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | 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 / Humidity: 75 % / Chamber temperature: 277 K |
-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: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 67.68 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software | Name: RELION / Version: 3.1.2 / Category: 3D reconstruction | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75000 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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