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Yorodumi- PDB-8ea4: V-K CAST Transpososome from Scytonema hofmanni, minor configuration -
+Open data
-Basic information
Entry | Database: PDB / ID: 8ea4 | |||||||||
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Title | V-K CAST Transpososome from Scytonema hofmanni, minor configuration | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN/RNA/DNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Function / homology | Function and homology information rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / cytoplasm Similarity search - Function | |||||||||
Biological species | Scytonema hofmannii (bacteria) Escherichia coli (E. coli) synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | |||||||||
Authors | Rizo, A.R. / Park, J.-U. / Tsai, A.W. / Kellogg, E.K. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Nature / Year: 2023 Title: Structures of the holo CRISPR RNA-guided transposon integration complex. Authors: Jung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg / Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ea4.cif.gz | 1.3 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8ea4.ent.gz | 996.4 KB | Display | PDB format |
PDBx/mmJSON format | 8ea4.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8ea4_validation.pdf.gz | 2.3 MB | Display | wwPDB validaton report |
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Full document | 8ea4_full_validation.pdf.gz | 2.5 MB | Display | |
Data in XML | 8ea4_validation.xml.gz | 196.3 KB | Display | |
Data in CIF | 8ea4_validation.cif.gz | 293.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ea/8ea4 ftp://data.pdbj.org/pub/pdb/validation_reports/ea/8ea4 | HTTPS FTP |
-Related structure data
Related structure data | 27972MC 7svuC 8ea3C C: citing same article (ref.) M: map data used to model this data |
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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 , 5 types, 24 molecules ABCDEFGHIJKLMOQSWXYZwxyz
#1: Protein | Mass: 31444.617 Da / Num. of mol.: 13 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL3 #2: Protein | | Mass: 73280.719 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: A0A8M0FGU0 #3: 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 #4: Protein | | Mass: 10290.816 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsO, HMPREF9346_03742 / Production host: Escherichia coli (E. coli) / References: UniProt: D8EB41 #5: Protein | Mass: 66637.070 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) |
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-DNA chain , 6 types, 6 molecules 123456
#6: DNA chain | Mass: 43440.840 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#7: DNA chain | Mass: 15775.137 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#8: DNA chain | Mass: 21814.982 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#9: DNA chain | Mass: 23080.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#10: DNA chain | Mass: 15443.954 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#11: DNA chain | Mass: 6086.917 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-RNA chain , 1 types, 1 molecules 7
#12: RNA chain | Mass: 85261.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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-Non-polymers , 2 types, 28 molecules
#13: Chemical | ChemComp-MG / #14: Chemical | ChemComp-ATP / |
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-Details
Has ligand of interest | N |
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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: Sh_CAST Transpososome / Type: COMPLEX / Entity ID: #1-#12 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: [Scytonema hofmanni] UTEX 2349 (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | 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: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67096 / Symmetry type: POINT |