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- PDB-7qd6: Cryo-EM structure of Tn4430 TnpA transposase from Tn3 family in c... -

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Basic information

Entry
Database: PDB / ID: 7qd6
TitleCryo-EM structure of Tn4430 TnpA transposase from Tn3 family in complex with strand-transfer like DNA product
Components
  • (IR71st transferred strand) x 2
  • IR71st non transferred strand
  • Transposase for transposon Tn4430
KeywordsRECOMBINATION / DNA transposition / Tn3 family / antibiotic resistance / protein metamorphosis
Function / homology
Function and homology information


transposase activity / DNA transposition / DNA binding
Similarity search - Function
Tn3 transposase DDE domain / Domain of unknown function DUF4158 / : / Tn3 transposase DDE domain / Domain of unknown function (DUF4158)
Similarity search - Domain/homology
DNA / DNA (> 10) / Transposase for transposon Tn4430
Similarity search - Component
Biological speciesBacillus thuringiensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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
Research Foundation - Flanders (FWO)G054617N
Fonds National de la Recherche Scientifique (FRNS)J.0162.16 Belgium
Fonds National de la Recherche Scientifique (FRNS)J.0096.20 Belgium
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: IR71st non transferred strand
C: IR71st transferred strand
G: IR71st transferred strand
D: Transposase for transposon Tn4430
E: IR71st non transferred strand
F: IR71st transferred strand
H: IR71st transferred strand


Theoretical massNumber of molelcules
Total (without water)340,1668
Polymers340,1668
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transposase for transposon Tn4430


Mass: 116992.117 Da / Num. of mol.: 2 / Mutation: S911R
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
#2: DNA chain IR71st non transferred strand


Mass: 21858.010 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Bacillus thuringiensis (bacteria)
#3: DNA chain IR71st transferred strand


Mass: 24127.393 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Bacillus thuringiensis (bacteria)
#4: DNA chain IR71st transferred strand


Mass: 7105.598 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Bacillus thuringiensis (bacteria)

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1TnpA-IR71st complexCOMPLEXhyperactive TnpA mutant S911R in complex with 71 bp branched DNA substrate containing trasnposon recognition sequence.#1-#20MULTIPLE SOURCES
2hyperactive TnpA mutant S911RCOMPLEXhyperactive TnpA mutant S911R#11RECOMBINANT
3DNA substrateCOMPLEXDNA substrate#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Bacillus thuringiensis (bacteria)1428
23Bacillus thuringiensis (bacteria)1428
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
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 / Humidity: 90 %

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3200 nm / Nominal defocus min: 400 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 3 sec. / Electron dose: 62 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4155
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.1.0particle selection
2SerialEM3.0.8image acquisition
4CTFFIND4CTF correction
7Coot0.9.5model fitting
9RELION3.1.2initial Euler assignment
10RELION3.1.2final Euler assignment
11RELION3.1.0classification
12RELION3.1.23D reconstruction
13PHENIX1.19.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 884091 / Details: cryoSPARC blob picker
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139519 / Symmetry type: POINT
Atomic model buildingB value: 167.8 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00519944
ELECTRON MICROSCOPYf_angle_d0.88627882
ELECTRON MICROSCOPYf_dihedral_angle_d22.2877726
ELECTRON MICROSCOPYf_chiral_restr0.0483100
ELECTRON MICROSCOPYf_plane_restr0.0072804

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