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- PDB-6x68: Cryo-EM structure of piggyBac transposase synaptic complex with h... -

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Basic information

Entry
Database: PDB / ID: 6x68
TitleCryo-EM structure of piggyBac transposase synaptic complex with hairpin DNA (SNHP)
Components
  • Transposase
  • hairpin DNA
KeywordsDNA BINDING PROTEIN/DNA / piggyBac / synaptic complex / hairpin DNA / transposase / DNA BINDING PROTEIN-DNA complex
Function / homologyPiggyBac transposable element-derived protein / Transposase IS4 / DNA / DNA (> 10) / Transposase
Function and homology information
Biological speciesTrichoplusia ni (cabbage looper)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsChen, Q. / Hickman, A.B. / Dyda, F.
Funding support United States, 1items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 22, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-22073
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
C: Transposase
D: Transposase
A: hairpin DNA
B: hairpin DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,07512
Polymers181,6534
Non-polymers4228
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15380 Å2
ΔGint-112 kcal/mol
Surface area61870 Å2

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Components

#1: Protein Transposase


Mass: 68012.969 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichoplusia ni (cabbage looper) / Cell line (production host): EXPI293F / Production host: Homo sapiens (human) / References: UniProt: Q283G1
#2: DNA chain hairpin DNA


Mass: 22813.643 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Trichoplusia ni (cabbage looper)
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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

ComponentName: piggyBac transposase complex with hairpin DNA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Trichoplusia ni (cabbage looper)
Source (recombinant)Organism: Homo sapiens (human) / Cell: EXPI293F
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2200 mMsodium chlorideNaCl1
310 mMcalcium chlorideCaCl21
41 mMTCEP1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: METHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 10 sec. / Electron dose: 73.7 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEM3.5image acquisition
4RELION3CTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13Rosettamodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2623168
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35960 / Symmetry type: POINT
Atomic model buildingB value: 109 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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