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Yorodumi- PDB-8ff4: Cryo-EM structure of Cascade-DNA-TniQ-TnsC complex (composite) in... -
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-Basic information
Entry | Database: PDB / ID: 8ff4 | ||||||
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Title | Cryo-EM structure of Cascade-DNA-TniQ-TnsC complex (composite) in type I-B CAST system | ||||||
Components |
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Keywords | DNA BINDING PROTEIN / CRISPR / transposon / TnsC / TniQ | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å | ||||||
Authors | Chang, L. / Wang, S. | ||||||
Funding support | United States, 1items
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Citation | Journal: Cell / Year: 2023 Title: Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector. Authors: Shukun Wang / Clinton Gabel / Romana Siddique / Thomas Klose / Leifu Chang / Abstract: Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key ...Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ff4.cif.gz | 1.1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8ff4.ent.gz | 911 KB | Display | PDB format |
PDBx/mmJSON format | 8ff4.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ff/8ff4 ftp://data.pdbj.org/pub/pdb/validation_reports/ff/8ff4 | HTTPS FTP |
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-Related structure data
Related structure data | 29039MC 8fcjC 8fcuC 8fcvC 8fcwC 8fcxC 8fd2C 8fd3C 8ff5C 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
-Type I-B CRISPR-associated protein ... , 5 types, 12 molecules ABCDEFGHIJKL
#1: Protein | Mass: 24852.906 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: cas5 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IG00 | ||||
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#2: Protein | Mass: 24945.744 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: cas6 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IH92 | ||||
#3: Protein | Mass: 37298.996 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: CDG76_09080 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IG15 #4: Protein | | Mass: 63474.328 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: CDG76_09085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IGR9 #5: Protein | Mass: 16314.365 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: CDG76_09085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IGR9 |
-RNA chain , 1 types, 1 molecules M
#6: RNA chain | Mass: 22876.527 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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-DNA chain , 2 types, 2 molecules NO
#7: DNA chain | Mass: 26108.730 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#8: DNA chain | Mass: 26313.898 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein , 2 types, 8 molecules PQRSTUVW
#9: Protein | Mass: 37591.961 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: CDG76_09005 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IFP5 |
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#10: Protein | Mass: 43521.934 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) Gene: CDG76_08990 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IFM2 |
-Non-polymers , 2 types, 14 molecules
#11: Chemical | ChemComp-ATP / #12: Chemical | ChemComp-MG / |
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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: Structure of Cascade-DNA-TniQ-TnsC complex (composite) in type I-B CAST system Type: COMPLEX / Entity ID: #1-#10 / Source: MULTIPLE SOURCES | ||||||||||||
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Source (natural) |
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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 | 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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING ONLY |
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3D reconstruction | Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 281621 / Symmetry type: POINT |