+Open data
-Basic information
Entry | Database: PDB / ID: 7n6i | ||||||
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Title | ATP-bound TnsC-TniQ complex from ShCAST system | ||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / CRISPR / Transposition / AAA+ ATPase / Tn7 / DNA BINDING PROTEIN-DNA complex | ||||||
Function / homology | ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) Function and homology information | ||||||
Biological species | Scytonema hofmannii (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Park, J. / Tsai, A.W.L. / Mehrotra, E. / Kellogg, E.H. | ||||||
Funding support | United States, 1items
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Citation | Journal: Science / Year: 2021 Title: Structural basis for target site selection in RNA-guided DNA transposition systems. Authors: Jung-Un Park / Amy Wei-Lun Tsai / Eshan Mehrotra / Michael T Petassi / Shan-Chi Hsieh / Ailong Ke / Joseph E Peters / Elizabeth H Kellogg / Abstract: CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron ...CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications. | ||||||
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 | 7n6i.cif.gz | 411.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7n6i.ent.gz | 322.9 KB | Display | PDB format |
PDBx/mmJSON format | 7n6i.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7n6i_validation.pdf.gz | 1.7 MB | Display | wwPDB validaton report |
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Full document | 7n6i_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 7n6i_validation.xml.gz | 77.7 KB | Display | |
Data in CIF | 7n6i_validation.cif.gz | 108.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/n6/7n6i ftp://data.pdbj.org/pub/pdb/validation_reports/n6/7n6i | HTTPS FTP |
-Related structure data
Related structure data | 23726MC 7m99C 7m9aC 7m9bC 7m9cC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 2 types, 10 molecules ABCDEFGHIJ
#1: Protein | Mass: 19011.240 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli BL21 (bacteria) #2: Protein | Mass: 31444.617 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli BL21 (bacteria) |
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-DNA chain , 2 types, 2 molecules KL
#3: DNA chain | Mass: 5126.320 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 5279.559 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 2 types, 14 molecules
#5: Chemical | ChemComp-ATP / #6: Chemical | ChemComp-MG / |
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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: ATP-bound TnsC-TniQ complex from ShCAST system / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.4 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Scytonema hofmannii (bacteria) | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21 (bacteria) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Excess of TniQ was added to ATP-bound TnsC filaments | |||||||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R2/2 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot for 7 seconds before plunging |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 63000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4172 |
Image scans | Width: 5760 / Height: 4092 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 648550 / Details: Neural net based particle picking | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34546 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT Details: Homology model was built using the crystal structure of TniQ (PDB:6V9P) by GalaxyTBM server. This model is manually docked into the cryo-EM density using UCSF Chimera. | ||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6V9P Pdb chain-ID: A / Accession code: 6V9P / Source name: PDB / Type: experimental model |