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- EMDB-22073: Cryo-EM structure of piggyBac transposase synaptic complex with h... -

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Basic information

Entry
Database: EMDB / ID: EMD-22073
TitleCryo-EM structure of piggyBac transposase synaptic complex with hairpin DNA (SNHP)
Map data
Sample
  • Complex: piggyBac transposase complex with hairpin DNA
    • Protein or peptide: Transposase
    • DNA: hairpin DNA
  • Ligand: CALCIUM IONCalcium
  • Ligand: ZINC ION
KeywordspiggyBac / synaptic complex / hairpin DNA / transposase / DNA BINDING PROTEIN-DNA complex
Function / homologyPiggyBac transposable element-derived protein / Transposase IS4 / Transposase
Function and homology information
Biological speciesTrichoplusia ni (cabbage looper)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsChen Q / Hickman AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK093660 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis of seamless excision and specific targeting by piggyBac transposase.
Authors: Qiujia Chen / Wentian Luo / Ruth Ann Veach / Alison B Hickman / Matthew H Wilson / Fred Dyda /
Abstract: The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates ...The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.
History
DepositionMay 27, 2020-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x68
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22073.png

Downloads & links

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Map

FileDownload / File: emd_22073.map.gz / Format: CCP4 / Size: 26.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.05813726 - 0.07750554
Average (Standard dev.)0.000006409101 (±0.0027888021)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions190190190
Spacing190190190
CellA=B=C: 205.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z190190190
origin x/y/z0.0000.0000.000
length x/y/z205.200205.200205.200
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS190190190
D min/max/mean-0.0580.0780.000

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Supplemental data

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Sample components

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Entire : piggyBac transposase complex with hairpin DNA

EntireName: piggyBac transposase complex with hairpin DNA
Components
  • Complex: piggyBac transposase complex with hairpin DNA
    • Protein or peptide: Transposase
    • DNA: hairpin DNA
  • Ligand: CALCIUM IONCalcium
  • Ligand: ZINC ION

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Supramolecule #1: piggyBac transposase complex with hairpin DNA

SupramoleculeName: piggyBac transposase complex with hairpin DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Trichoplusia ni (cabbage looper)

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Macromolecule #1: Transposase

MacromoleculeName: Transposase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 68.012969 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG SEILDEQNVI EQPGSSLASN KILTLPQRT IRGKNKHCWS TSKSTRRSRV SALNIVRSQR GPTRMCRNIY DPLLCFKLFF TDEIISEIVK WTNAEISLKR R ESMTGATF ...String:
MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG SEILDEQNVI EQPGSSLASN KILTLPQRT IRGKNKHCWS TSKSTRRSRV SALNIVRSQR GPTRMCRNIY DPLLCFKLFF TDEIISEIVK WTNAEISLKR R ESMTGATF RDTNEDEIYA FFGILVMTAV RKDNHMSTDD LFDRSLSMVY VSVMSRDRFD FLIRCLRMDD KSIRPTLREN DV FTPVRKI WDLFIHQCIQ NYTPGAHLTI DEQLLGFRGR CPFRMYIPNK PSKYGIKILM MCDSGTKYMI NGMPYLGRGT QTN GVPLGE YYVKELSKPV RGSCRNITCD NWFTSIPLAK NLLQEPYKLT IVGTVRSNKR EIPEVLKNSR SRPVGTSMFC FDGP LTLVS YKPKPAKMVY LLSSCDEDAS INESTGKPQM VMYYNQTKGG VDTLDQMCSV MTCSRKTNRW PMALLYGMIN IACIN SFII YSHNVSSKGE KVQSRKKFMR NLYMSLTSSF MRKRLEAPTL KRYLRDNISN ILPNEVPGTS DDSTEEPVTK KRTYCT YCP SKIRRKANAS CKKCKKVICR EHNIDMCQSC F

UniProtKB: Transposase

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Macromolecule #2: hairpin DNA

MacromoleculeName: hairpin DNA / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 22.813643 KDa
SequenceString: (DC)(DA)(DT)(DG)(DC)(DG)(DT)(DC)(DA)(DA) (DT)(DT)(DT)(DT)(DA)(DC)(DG)(DC)(DA)(DG) (DA)(DC)(DT)(DA)(DT)(DC)(DT)(DT)(DT) (DC)(DT)(DA)(DG)(DG)(DG)(DT)(DT)(DA)(DA) (DC) (DC)(DC)(DT)(DA)(DG)(DA) ...String:
(DC)(DA)(DT)(DG)(DC)(DG)(DT)(DC)(DA)(DA) (DT)(DT)(DT)(DT)(DA)(DC)(DG)(DC)(DA)(DG) (DA)(DC)(DT)(DA)(DT)(DC)(DT)(DT)(DT) (DC)(DT)(DA)(DG)(DG)(DG)(DT)(DT)(DA)(DA) (DC) (DC)(DC)(DT)(DA)(DG)(DA)(DA)(DA) (DG)(DA)(DT)(DA)(DG)(DT)(DC)(DT)(DG)(DC) (DG)(DT) (DA)(DA)(DA)(DA)(DT)(DT)(DG) (DA)(DC)(DG)(DC)(DA)(DT)(DG)

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris-HClTris
200.0 mMsodium chlorideNaClSodium chloride
10.0 mMcalcium chlorideCaCl2
1.0 mMTCEP
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: METHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 10.0 sec. / Average electron dose: 73.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2623168
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 35960

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 109 / Target criteria: Correlation coefficient
Output model

PDB-6x68:
Cryo-EM structure of piggyBac transposase synaptic complex with hairpin DNA (SNHP)

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