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- PDB-8ff5: Cryo-EM structure of Cascade-DNA-fullRloop in type I-B CAST system -
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Open data
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Basic information
Entry | Database: PDB / ID: 8ff5 | ||||||
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Title | Cryo-EM structure of Cascade-DNA-fullRloop in type I-B CAST system | ||||||
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![]() | DNA BINDING PROTEIN / CRISPR / DNA recognition | ||||||
Function / homology | ![]() | ||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å | ||||||
![]() | Chang, L. / Wang, S. | ||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 606.8 KB | Display | ![]() |
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PDB format | ![]() | 490.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 29040MC ![]() 8fcjC ![]() 8fcuC ![]() 8fcvC ![]() 8fcwC ![]() 8fcxC ![]() 8fd2C ![]() 8fd3C ![]() 8ff4C M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Type I-B CRISPR-associated protein ... , 4 types, 11 molecules BCDEFGHIJKL
#2: Protein | Mass: 24945.744 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: cas6 / Production host: ![]() ![]() | ||||
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#3: Protein | Mass: 37298.996 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: CDG76_09080 / Production host: ![]() ![]() #4: Protein | | Mass: 63474.328 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: CDG76_09085 / Production host: ![]() ![]() #5: Protein | Mass: 16314.365 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: CDG76_09085 / Production host: ![]() ![]() |
-DNA chain , 2 types, 2 molecules NO
#7: DNA chain | Mass: 19913.799 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#8: DNA chain | Mass: 20149.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein / RNA chain , 2 types, 2 molecules AM
#1: Protein | Mass: 24852.906 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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#6: RNA chain | Mass: 22876.527 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 |
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Sample preparation
Component | Name: Ternary complex of Cascade proteins, crRNA and target DNA with the complete R loop formed. Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES | ||||||||||||
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Source (natural) |
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Source (recombinant) | Organism: ![]() ![]() | ||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Vitrification | Cryogen name: ETHANE |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / 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) |
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Processing
CTF correction | Type: PHASE FLIPPING ONLY |
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3D reconstruction | Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 116526 / Symmetry type: POINT |