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- PDB-6pqu: Cryo-EM structure of HzTransib/nicked TIR substrate DNA pre-react... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6pqu | ||||||
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Title | Cryo-EM structure of HzTransib/nicked TIR substrate DNA pre-reaction complex (PRC) | ||||||
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![]() | recombination/dna / RAG-like transposase / DDE family enzyme / Transib / Terminal inverted repeat. / RECOMBINATION / recombination-dna complex | ||||||
Function / homology | metal ion binding / : / DNA / DNA (> 10) / Putative DNA-mediated transposase![]() | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | ||||||
![]() | Liu, C. / Yang, Y. / Schatz, D.G. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structures of a RAG-like transposase during cut-and-paste transposition. Authors: Chang Liu / Yang Yang / David G Schatz / ![]() Abstract: Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed ...Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed vertebrates. Here we report one crystal structure and five cryo-electron microscopy structures of Transib, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase. | ||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 237.2 KB | Display | ![]() |
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PDB format | ![]() | 180.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 20453MC ![]() 6pqnC ![]() 6pqrC ![]() 6pqxC ![]() 6pqyC ![]() 6pr5C C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 1 types, 2 molecules AE
#1: Protein | Mass: 56582.734 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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-DNA chain , 3 types, 6 molecules BFCGDH
#2: DNA chain | Mass: 4889.177 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) ![]() #3: DNA chain | Mass: 4956.244 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) ![]() #4: DNA chain | Mass: 9792.304 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) ![]() |
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-Non-polymers , 3 types, 6 molecules ![](data/chem/img/MG.gif)
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#5: Chemical | #6: Chemical | #7: Chemical | |
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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 |
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Sample preparation
Component | Name: Pre-reaction complex of HzTransib with nicked TIR substrate DNA Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: Solutions were made fresh from concentrated and filtered to avoid microbial contamination. | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Recombinantly expressed HzTransib transposase was mixed with chemically synthesized TIR substrate DNA. The complex was further purified on size-exclusion chromatography column. The final ...Details: Recombinantly expressed HzTransib transposase was mixed with chemically synthesized TIR substrate DNA. The complex was further purified on size-exclusion chromatography column. The final complex was monodisperse. | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 296 K / Details: Blot for 3 seconds before plunging |
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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 / Details: Preliminary grid screening was performed manually. |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 8 sec. / Electron dose: 52.2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) Details: Images were collected in movie-mode at 5 frames per second. |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
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Processing
Software | Name: PHENIX / Version: 1.15.2_3472: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59333 / Num. of class averages: 10 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient Details: Initial local fitting was done using UCSF Chimera, then manually adjusted and rebuilt in Coot. Final model was refined using Phenix real-space refinement. | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6PQN Pdb chain-ID: A / Accession code: 6PQN / Pdb chain residue range: 21-501 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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