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- PDB-6pqy: Cryo-EM structure of HzTransib/TIR DNA transposon end complex (TEC) -

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Basic information

Entry
Database: PDB / ID: 6pqy
TitleCryo-EM structure of HzTransib/TIR DNA transposon end complex (TEC)
Components
  • DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
  • DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*G)-3')
  • Putative DNA-mediated transposase
KeywordsRECOMBINATION / RAG-like transposase / DDE family enzyme / Transib / Terminal inverted repeat.
Function / homologymetal ion binding / DNA / DNA (> 10) / Putative DNA-mediated transposase
Function and homology information
Biological speciesHelicoverpa zea (corn earworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsLiu, C. / Yang, Y. / Schatz, D.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI137079 United States
CitationJournal: Nature / Year: 2019
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
DepositionJul 10, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 4, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Putative DNA-mediated transposase
B: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
C: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*G)-3')
D: Putative DNA-mediated transposase
E: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
F: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*G)-3')


Theoretical massNumber of molelcules
Total (without water)132,7586
Polymers132,7586
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The whole assembly was purified by gel filtration. A protein standard containing 6 different protein ranging from 1.7 kDa to 670 kDa was also passed through the same gel ...Evidence: gel filtration, The whole assembly was purified by gel filtration. A protein standard containing 6 different protein ranging from 1.7 kDa to 670 kDa was also passed through the same gel filtration column. Comparison of the two chromatography profiles indicate the assembly has an approximate molecular mass of 220-250 kDa, which is about the same as the combined molecular weight of two copies of HzTransib transposase and two TIR substrate DNA.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Putative DNA-mediated transposase


Mass: 56582.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicoverpa zea (corn earworm) / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: B0F0C5
#2: DNA chain DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')


Mass: 4956.244 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)
#3: DNA chain DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*G)-3')


Mass: 4840.140 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Transposon end complex of HzTransib with cleaved TIR substrate DNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Helicoverpa zea (corn earworm)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9
Buffer solutionpH: 7.5
Details: Solutions were made fresh from concentrated and filtered to avoid microbial contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisNH2C(CH2OH)31
250 mMpotassium chlorideKCl1
310 mMMagnesium chlorideMgCl21
41 mMTris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 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 reaction was carried out at 30 C for 1h, and the complex was further purified on ...Details: Recombinantly expressed HzTransib transposase was mixed with chemically synthesized TIR substrate DNA. The reaction was carried out at 30 C for 1h, and the complex was further purified on size-exclusion chromatography column. The final complex was monodisperse.
Specimen supportDetails: unspecified
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 54.4 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 opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEMimage acquisition
4RELION3CTF correction
7UCSF Chimera1.13model fitting
9PHENIX1.15model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26397 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: 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 buildingPDB-ID: 6PQN

6pqn


Pdb chain-ID: A / Accession code: 6PQN / Pdb chain residue range: 21-501 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0078988
ELECTRON MICROSCOPYf_angle_d0.96212400
ELECTRON MICROSCOPYf_dihedral_angle_d18.3745236
ELECTRON MICROSCOPYf_chiral_restr0.0631400
ELECTRON MICROSCOPYf_plane_restr0.0211380

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