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- EMDB-13908: Cryo-EM structure of Tn4430 TnpA transposase from Tn3 family in c... -

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Basic information

Entry
Database: EMDB / ID: EMD-13908
TitleCryo-EM structure of Tn4430 TnpA transposase from Tn3 family in complex with 48 bp long transposon end DNA
Map datacomposite map of consensus and multibody refinement used for model refinement
Sample
  • Complex: TnpA-IR48 complex
    • Complex: hyperactive TnpA mutant S911R
      • Protein or peptide: Transposase for transposon Tn4430
    • Complex: DNA substrate
      • DNA: IR48 DNA substrate, non transferred strand
      • DNA: IR48 transferred strand
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsShkumatov AV / Oger CA / Aryanpour N / Hallet BF / Efremov RG
Funding support Belgium, 4 items
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, 2021-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13908.png

Downloads & links

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Map

FileDownload / File: emd_13908.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map of consensus and multibody refinement used for model refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.76 Å/pix.
x 440 pix.
= 336.16 Å		0.76 Å/pix.
x 440 pix.
= 336.16 Å		0.76 Å/pix.
x 440 pix.
= 336.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.764 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.8
Minimum - Maximum-43.462326 - 76.96004
Average (Standard dev.)0.00032991404 (±1.000834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 336.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13908_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_13908_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_13908_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TnpA-IR48 complex

EntireName: TnpA-IR48 complex
Components
  • Complex: TnpA-IR48 complex
    • Complex: hyperactive TnpA mutant S911R
      • Protein or peptide: Transposase for transposon Tn4430
    • Complex: DNA substrate
      • DNA: IR48 DNA substrate, non transferred strand
      • DNA: IR48 transferred strand

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Supramolecule #1: TnpA-IR48 complex

SupramoleculeName: TnpA-IR48 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: hyperactive TnpA mutant S911R in complex with 48 bp DNA containing transposon recognition sequence.

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Supramolecule #2: hyperactive TnpA mutant S911R

SupramoleculeName: hyperactive TnpA mutant S911R / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: hyperactive TnpA mutant S911R
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA substrate

SupramoleculeName: DNA substrate / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 / Details: DNA substrate
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Transposase for transposon Tn4430

MacromoleculeName: Transposase for transposon Tn4430 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Molecular weightTheoretical: 116.992117 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGVKQLLSEA QRNELMDLSR LTEWDLVTFH TFSKHDLHLI LKHRRGYNRL GFALQLVLIR YPGWSLTEYK DIPQYVVAYV ASQLQIPPE EFLVYAKRGN TLWEHLGEIR TEYGYQNFSS EYKETLLQFL VQQAMDNNNT LYLIEITIST LRKMKVILPA M YVIEDIVW ...String:
MGVKQLLSEA QRNELMDLSR LTEWDLVTFH TFSKHDLHLI LKHRRGYNRL GFALQLVLIR YPGWSLTEYK DIPQYVVAYV ASQLQIPPE EFLVYAKRGN TLWEHLGEIR TEYGYQNFSS EYKETLLQFL VQQAMDNNNT LYLIEITIST LRKMKVILPA M YVIEDIVW EAKQQADQKV YSILHDGLVQ EQKDQLDALL LPTINGKSPL AWLKDVPAQP SPESFLKVID RLQFVQKIGL TI DTTKINT NRLRQLARLG SKYEPYAFRR FNEVKRYSML VSFLLEITQD LIDYAIEIHD RLMMNLQTKG KKEQDEIQQA NGK KLNEKI LQFITVCGTL IEAKETGKDA FAALDEVMSW NEMVESVEEA KQLSRPLNYD YLDLLNTRYS YVRRYAPTLL RSLH FRATK SGEPVLQALD TIHELNETGK RKVPHGAPLH FVSNRWQKHV YDDDGNINRH YYELAALTEL RNHIRSGDIF VSGSR HHKA FDDYLIPYDE WNEVSNIPNG LTAPLKAEDY ITDRINRLNE HLEWLSKNSE KLEGVDISQG KLHVERLDRG TPEEAK AFS KLLHSMLPRI KLTDLLIEVA SWTGFHDQFI HASTNQSPDQ EEQNIVLATL MAMGTNIGLT KMAEATPGIS YRQMANA SQ WRMYDDAMVR AQSILVNFQK EQKLSSYWGD GTTSSSDGMR LSIAVRSLHA DSNPHYGTGK GGTIYRFVSD QLSAYHVK V ITTNARDALH VLDGLLHHET DLKIEEHYTD TAGYTDQVFA LTHLLGFRFA PRIRDLADTK LFSIPGGEEY ENVQALLKG KINVKLIKEN YEDIRRLAYS VQTGKVSSAL IMGKLGSYAR QNKLATALGE MGRIEKTLFT LDYISNKAVR RRVQKGLNKG EAINALARI IFFGQRGEFR ERALQDQLQR ARALNIIINA ISVWNTVYME KAVEELKARG EFREDLMPYA WPLGWEHINF L GEYKFEGL HDTGQMNLRP LRIKEPFYSP IRSFLEQKLI SEEDLNSAVD HHHHHH

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Macromolecule #2: IR48 DNA substrate, non transferred strand

MacromoleculeName: IR48 DNA substrate, non transferred strand / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Molecular weightTheoretical: 14.756492 KDa
SequenceString:
(DC)(DC)(DA)(DT)(DG)(DG)(DG)(DG)(DG)(DT) (DA)(DC)(DC)(DG)(DC)(DC)(DA)(DG)(DC)(DA) (DT)(DT)(DT)(DC)(DG)(DG)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DC)(DC)(DA)(DC)(DG)(DC) (DT) (DA)(DA)(DG)(DA)(DT)(DC)(DC)(DT)

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Macromolecule #3: IR48 transferred strand

MacromoleculeName: IR48 transferred strand / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Molecular weightTheoretical: 14.813468 KDa
SequenceString:
(DA)(DG)(DG)(DA)(DT)(DC)(DT)(DT)(DA)(DG) (DC)(DG)(DT)(DG)(DG)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DC)(DC)(DG)(DA)(DA)(DA)(DT) (DG)(DC)(DT)(DG)(DG)(DC)(DG)(DG)(DT)(DA) (DC) (DC)(DC)(DC)(DC)(DA)(DT)(DG)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES (pH 7.5), 100 mM NaCl, 30 mM L-Arg HCL
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 %

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.7 µm / Nominal defocus min: 0.6 µm
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 3 / Number real images: 11764 / Average exposure time: 3.0 sec. / Average electron dose: 62.0 e/Å2

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Image processing

Particle selectionNumber selected: 2635462 / Details: cryoSPARC blob picker
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER / Details: ab initio model from cryoSPARC
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 478011
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 12.4
Output model

PDB-7qd5:
Cryo-EM structure of Tn4430 TnpA transposase from Tn3 family in complex with 48 bp long transposon end DNA

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