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- PDB-7qd8: Cryo-EM structure of Tn4430 TnpA transposase from Tn3 family in a... -

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Basic information

Entry
Database: PDB / ID: 7qd8
TitleCryo-EM structure of Tn4430 TnpA transposase from Tn3 family in apo state
ComponentsTransposase for transposon Tn4430
KeywordsRECOMBINATION / DNA transposition / Tn3 family / antibiotic resistance / protein metamorphosis
Function / homologyTn3 transposase DDE domain / Domain of unknown function DUF4158 / : / Tn3 transposase DDE domain / Domain of unknown function (DUF4158) / transposase activity / DNA transposition / DNA binding / Transposase for transposon Tn4430
Function and homology information
Biological speciesBacillus thuringiensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsShkumatov, A.V. / Oger, C.A. / Aryanpour, N. / Hallet, B.F. / Efremov, R.G.
Funding support Belgium, 4items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
Fonds National de la Recherche Scientifique (FNRS)J.0162.16 Belgium
Fonds National de la Recherche Scientifique (FNRS)J.0096.20
CitationJournal: Nat Commun / Year: 2022
Title: Structural insight into Tn3 family transposition mechanism.
Authors: Alexander V Shkumatov / Nicolas Aryanpour / Cédric A Oger / Gérôme Goossens / Bernard F Hallet / Rouslan G Efremov /
Abstract: Transposons are diverse mobile genetic elements that play the critical role as genome architects in all domains of life. Tn3 is a widespread family and among the first identified bacterial ...Transposons are diverse mobile genetic elements that play the critical role as genome architects in all domains of life. Tn3 is a widespread family and among the first identified bacterial transposons famed for their contribution to the dissemination of antibiotic resistance. Transposition within this family is mediated by a large TnpA transposase, which facilitates both transposition and target immunity. Howtever, a structural framework required for understanding the mechanism of TnpA transposition is lacking. Here, we describe the cryo-EM structures of TnpA from Tn4430 in the apo form and paired with transposon ends before and after DNA cleavage and strand transfer. We show that TnpA has an unusual architecture and exhibits a family specific regulatory mechanism involving metamorphic refolding of the RNase H-like catalytic domain. The TnpA structure, constrained by a double dimerization interface, creates a peculiar topology that suggests a specific role for the target DNA in transpososome assembly and activation.
History
DepositionNov 26, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transposase for transposon Tn4430
B: Transposase for transposon Tn4430


Theoretical massNumber of molelcules
Total (without water)233,8442
Polymers233,8442
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6240 Å2
ΔGint-45 kcal/mol
Surface area84730 Å2
MethodPISA

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Components

#1: Protein Transposase for transposon Tn4430


Mass: 116922.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus thuringiensis (bacteria) / Gene: tnpA / Production host: Escherichia coli (E. coli) / References: UniProt: P10021

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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: TnpA transposase in apo state / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 50 mM HEPES (pH 7.5), 100 mM NaCl, 30 mM L-Arg HCL
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5300 nm / Nominal defocus min: 600 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 10 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3724
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPU1image acquisition11
4CTFFIND4CTF correction
7Coot0.9.5model fitting
9PHENIX1.19.1model refinement
10cryoSPARC3.1.0initial Euler assignment
11cryoSPARC3.1.0final Euler assignment
12cryoSPARC3.1.0classification
13cryoSPARC3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4751313 / Details: cryoSPARC blob picker
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 399477 / Symmetry type: POINT
Atomic model buildingB value: 159 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414372
ELECTRON MICROSCOPYf_angle_d0.72619424
ELECTRON MICROSCOPYf_dihedral_angle_d4.8881898
ELECTRON MICROSCOPYf_chiral_restr0.0472160
ELECTRON MICROSCOPYf_plane_restr0.0052476

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