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- PDB-7lcc: Helitron transposase bound to LTS -

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Basic information

Entry
Database: PDB / ID: 7lcc
TitleHelitron transposase bound to LTS
Components
  • Helraiser K1068Q
  • LTS
KeywordsRECOMBINATION / Helitron / transposase / evolution / gene editing / gene capture / rolling circle / replication / nuclease / helicase / relaxase
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciessynthetic construct (others)
Myotis lucifugus (little brown bat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsKosek, D. / Dyda, F.
Funding support1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)
CitationJournal: Mol Cell / Year: 2021
Title: The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5'-transposon end.
Authors: Dalibor Kosek / Ivana Grabundzija / Haotian Lei / Ilija Bilic / Huaibin Wang / Yukun Jin / Graham F Peaslee / Alison B Hickman / Fred Dyda /
Abstract: Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle ...Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.
History
DepositionJan 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 1, 2021Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 3, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Helraiser K1068Q
B: LTS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)177,4773
Polymers177,4122
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Helraiser K1068Q


Mass: 171253.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Spodoptera frugiperda (fall armyworm)
#2: DNA chain LTS


Mass: 6158.058 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Myotis lucifugus (little brown bat)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / 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: Helraiser complex with LTS / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: YES
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / 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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 73.5 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEM3.5image acquisition
4RELION3CTF correction
9Rosettamodel refinement
10Coot9model refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1982458
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 937125 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: The initial model has been traced manualy, then refined in Rosetta and finished in Coot 9.0

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