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- EMDB-15213: Cryo-EM density map of Tn4430 TnpA hyperactive mutant (TnpA3X) in... -

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Basic information

Entry
Database: EMDB / ID: EMD-15213
TitleCryo-EM density map of Tn4430 TnpA hyperactive mutant (TnpA3X) in complex with IR48 substrate.
Map datafinal auto-sharpened map from Non-Uniform refinement in cryoSPARC v3.3.2 220518
Sample
  • Complex: TnpA3X-IR48 complex
    • Complex: TnpA 3X mutant
      • Protein or peptide: Transposase for transposon Tn4430
    • Complex: IR48 substrate
      • DNA: IR48 DNA substrate, non transferred strand
      • DNA: IR48 transferred strand
KeywordsTranposition / complex / hyperactive mutant / paired-end complex / DNA BINDING PROTEIN
Biological speciesBacillus thuringiensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsShkumatov AV / Liu Y / Efremov RG
Funding support Belgium, 1 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G.0266.15N Belgium
CitationJournal: Nucleic Acids Res / Year: 2023
Title: AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements.
Authors: Maricruz Fernandez / Alexander V Shkumatov / Yun Liu / Claire Stulemeijer / Sylvie Derclaye / Rouslan G Efremov / Bernard Hallet / David Alsteens /
Abstract: Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent ...Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent data on the structural architecture of the transposition complex, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds as a dimer to a single transposon end and then undergoes a structural transition that enables it to bind the second end cooperatively and to become activated for transposition catalysis, the latter step occurring at a much faster rate for the TnpA mutants. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.
History
DepositionJun 20, 2022-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateJun 21, 2023-
Current statusJun 21, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15213.png

Downloads & links

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Map

FileDownload / File: emd_15213.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal auto-sharpened map from Non-Uniform refinement in cryoSPARC v3.3.2 220518
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.57 Å/pix.
x 250 pix.
= 392. Å		1.57 Å/pix.
x 250 pix.
= 392. Å		1.57 Å/pix.
x 250 pix.
= 392. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.568 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-3.6764436 - 5.716514
Average (Standard dev.)-0.0031056753 (±0.12897083)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 392.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15213_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_15213_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_15213_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TnpA3X-IR48 complex

EntireName: TnpA3X-IR48 complex
Components
  • Complex: TnpA3X-IR48 complex
    • Complex: TnpA 3X mutant
      • Protein or peptide: Transposase for transposon Tn4430
    • Complex: IR48 substrate
      • DNA: IR48 DNA substrate, non transferred strand
      • DNA: IR48 transferred strand

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Supramolecule #1: TnpA3X-IR48 complex

SupramoleculeName: TnpA3X-IR48 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: hyperactive TnpA mutant (W24R, A174V, E740G) in complex with 48 bp DNA containing transposon recognition sequence.

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Supramolecule #2: TnpA 3X mutant

SupramoleculeName: TnpA 3X mutant / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: hyperactive TnpA mutant (W24R, A174V, E740G)
Source (natural)Organism: Bacillus thuringiensis (bacteria)

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Supramolecule #3: IR48 substrate

SupramoleculeName: IR48 substrate / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 / Details: DNA substrate
Source (natural)Organism: Bacillus thuringiensis (bacteria)

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Macromolecule #1: Transposase for transposon Tn4430

MacromoleculeName: Transposase for transposon Tn4430 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGVKQLLSEA QRNELMDLSR LTERDLVTFH TFSKHDLHLI LKHRRGYNRL GFALQLVLIR YPGWSLTEYK DIPQYVVAYV ASQLQIPPE EFLVYAKRGN TLWEHLGEIR TEYGYQNFSS EYKETLLQFL VQQAMDNNNT LYLIEITIST LRKMKVILPA M YVIEDIVW ...String:
MGVKQLLSEA QRNELMDLSR LTERDLVTFH TFSKHDLHLI LKHRRGYNRL GFALQLVLIR YPGWSLTEYK DIPQYVVAYV ASQLQIPPE EFLVYAKRGN TLWEHLGEIR TEYGYQNFSS EYKETLLQFL VQQAMDNNNT LYLIEITIST LRKMKVILPA M YVIEDIVW EAKQQVDQKV 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 VLDGLLHHGT DLKIEEHYTD TAGYTDQVFA LTHLLGFRFA PRIRDLADTK LFSIPGGEEY ENVQALLKG KINVKLIKEN YEDIRRLAYS VQTGKVSSAL IMGKLGSYAR QNKLATALGE MGRIEKTLFT LDYISNKAVR RRVQKGLNKG EAINALARI IFFGQRGEFR ERALQDQLQR ASALNIIINA 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 / Classification: DNA
Source (natural)Organism: Bacillus thuringiensis (bacteria)
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 / Classification: DNA
Source (natural)Organism: Bacillus thuringiensis (bacteria)
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

Concentration0.7 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES (pH 7.5), 100 mM NaCl, 30 mM L-Arg HCL
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.035 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3

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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: 4.0 µm / Nominal defocus min: 0.8 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) / Number grids imaged: 1 / Number real images: 6734 / Average exposure time: 3.0 sec. / Average electron dose: 64.6 e/Å2

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Image processing

Particle selectionNumber selected: 337079 / Details: CRYOLO v1.6.0
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: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. v3.3.2+220518)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 45955
FSC plot (resolution estimation)

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