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- EMDB-45082: piggyBat transposase protein-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-45082
TitlepiggyBat transposase protein-DNA complex
Map data
Sample
  • Complex: piggyBat-LE44
    • DNA: DNA (35-MER)
    • DNA: DNA (35-MER)
    • Protein or peptide: piggyBat transposase
  • Ligand: ZINC ION
Keywordstransposase / piggyBat / piggyBac-like element / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Biological speciesMyotis lucifugus (little brown bat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLannes L / Hickman AB / Dyda F
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
CitationJournal: Nat Commun / Year: 2025
Title: Activity of the mammalian DNA transposon piggyBat from Myotis lucifugus is restricted by its own transposon ends.
Authors: Alison B Hickman / Laurie Lannes / Christopher M Furman / Christina Hong / Lidiya Franklin / Rodolfo Ghirlando / Arpita Ghosh / Wentian Luo / Parthena Konstantinidou / Hernán A Lorenzi / ...Authors: Alison B Hickman / Laurie Lannes / Christopher M Furman / Christina Hong / Lidiya Franklin / Rodolfo Ghirlando / Arpita Ghosh / Wentian Luo / Parthena Konstantinidou / Hernán A Lorenzi / Anne Grove / Astrid D Haase / Matthew H Wilson / Fred Dyda /
Abstract: Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac-derived genes as ...Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac-derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties of piggyBat from Myotis lucifugus, the only known active mammalian DNA transposon, and show that its low activity in human cells is due to subterminal inhibitory DNA sequences. Activity can be dramatically improved by their removal, suggesting the existence of a mechanism for the suppression of transposon activity. The cryo-electron microscopy structure of the piggyBat transposase pre-synaptic complex showed an unexpected mode of DNA binding and recognition using C-terminal domains that are topologically different from those of the piggyBac transposase. Here we show that structure-based rational re-engineering of the transposase through the removal of putative phosphorylation sites and a changed domain organization - in combination with truncated transposon ends - results in a transposition system that is at least 100-fold more active than wild-type piggyBat.
History
DepositionMay 25, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45082.png

Downloads & links

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Map

FileDownload / File: emd_45082.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 240 pix.
= 206.4 Å		0.86 Å/pix.
x 240 pix.
= 206.4 Å		0.86 Å/pix.
x 240 pix.
= 206.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.00748902 - 0.019936305
Average (Standard dev.)0.00007779968 (±0.00094698474)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 206.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_45082_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45082_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : piggyBat-LE44

EntireName: piggyBat-LE44
Components
  • Complex: piggyBat-LE44
    • DNA: DNA (35-MER)
    • DNA: DNA (35-MER)
    • Protein or peptide: piggyBat transposase
  • Ligand: ZINC ION

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Supramolecule #1: piggyBat-LE44

SupramoleculeName: piggyBat-LE44 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Myotis lucifugus (little brown bat)

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Macromolecule #1: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Myotis lucifugus (little brown bat)
Molecular weightTheoretical: 10.868982 KDa
SequenceString:
(DC)(DA)(DC)(DT)(DT)(DG)(DG)(DA)(DT)(DT) (DG)(DC)(DG)(DG)(DG)(DA)(DA)(DA)(DC)(DG) (DA)(DG)(DT)(DT)(DA)(DA)(DG)(DT)(DC) (DG)(DG)(DC)(DT)(DC)(DG)

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Macromolecule #2: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Myotis lucifugus (little brown bat)
Molecular weightTheoretical: 10.668859 KDa
SequenceString:
(DC)(DG)(DA)(DG)(DC)(DC)(DG)(DA)(DC)(DT) (DT)(DA)(DA)(DC)(DT)(DC)(DG)(DT)(DT)(DT) (DC)(DC)(DC)(DG)(DC)(DA)(DA)(DT)(DC) (DC)(DA)(DA)(DG)(DT)(DG)

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Macromolecule #3: piggyBat transposase

MacromoleculeName: piggyBat transposase / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Myotis lucifugus (little brown bat)
Molecular weightTheoretical: 67.609297 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GGGGSGMAQH SDYSDDEFCA DKLSNYSCDS DLENASTSDE DSSDDEVMVR PRTLRRRRIS SSSSDSESDI EGGREEWSHV DNPPVLEDF LGHQGLNTDA VINNIEDAVK LFIGDDFFEF LVEESNRYYN QNRNNFKLSK KSLKWKDITP QEMKKFLGLI V LMGQVRKD ...String:
GGGGSGMAQH SDYSDDEFCA DKLSNYSCDS DLENASTSDE DSSDDEVMVR PRTLRRRRIS SSSSDSESDI EGGREEWSHV DNPPVLEDF LGHQGLNTDA VINNIEDAVK LFIGDDFFEF LVEESNRYYN QNRNNFKLSK KSLKWKDITP QEMKKFLGLI V LMGQVRKD RRDDYWTTEP WTETPYFGKT MTRDRFRQIW KAWHFNNNAD IVNESDRLCK VRPVLDYFVP KFINIYKPHQ QL SLDEGIV PWRGRLFFRV YNAGKIVKYG ILVRLLCESD TGYICNMEIY CGEGKRLLET IQTVVSPYTD SWYHIYMDNY YNS VANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG ETKFIRKNDI LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKK KIVKP NALIDYNKHM KGVDRADQYL SYYSILRRTV KWTKRLAMYM INCALFNSYA VYKSVRQRKM GFKMFLKQTA IHWLT DDIP EDMDIVPDLQ PVPSTSGMRA KPPTSDPPCR LSMDMRKHTL QAIVGSGKKK NILRRCRVCS VHKLRSETRY MCKFCN IPL HKGACFEKYH TLKNY

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 16.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 162244
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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