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- PDB-8sjd: Cryo-EM structure of the Hermes transposase bound to two right-en... -

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Basic information

Entry
Database: PDB / ID: 8sjd
TitleCryo-EM structure of the Hermes transposase bound to two right-ends of its DNA transposon.
Components
  • DNA (46-MER)
  • DNA (55-MER)
  • DNA (8-MER)
  • Hermes transposase
KeywordsRECOMBINATION/DNA / transposase / transpososome / BED domain / protein-DNA complex / RECOMBINATION-DNA complex
Function / homology
Function and homology information


protein dimerization activity / regulation of transcription by RNA polymerase II / DNA binding / nucleus / metal ion binding
Similarity search - Function
Hermes trasposase, DNA-binding domain / Hermes transposase DNA-binding domain / HAT, C-terminal dimerisation domain / : / hAT family C-terminal dimerisation region / BED zinc finger / Zinc finger, BED-type / Zinc finger BED-type profile. / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Hermes transposase
Similarity search - Component
Biological speciesMusca domestica (house fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsLannes, L. / Dyda, F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK036153-16 United States
CitationJournal: Nat Commun / Year: 2023
Title: Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding.
Authors: Laurie Lannes / Christopher M Furman / Alison B Hickman / Fred Dyda /
Abstract: The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo- ...The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end.
History
DepositionApr 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Hermes transposase
D: Hermes transposase
B: Hermes transposase
A: Hermes transposase
J: DNA (55-MER)
I: DNA (46-MER)
G: DNA (55-MER)
H: DNA (8-MER)
E: DNA (8-MER)
F: DNA (46-MER)


Theoretical massNumber of molelcules
Total (without water)347,38110
Polymers347,38110
Non-polymers00
Water00
1


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

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Components

#1: Protein
Hermes transposase


Mass: 70210.570 Da / Num. of mol.: 4 / Mutation: Q2E,K128G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Musca domestica (house fly) / Plasmid: pBAD/Myc-His / Details (production host): no fusion tag / Production host: Escherichia coli (E. coli) / Strain (production host): Top10 / References: UniProt: Q25438
#2: DNA chain DNA (55-MER)


Mass: 16909.779 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Musca domestica (house fly)
#3: DNA chain DNA (46-MER)


Mass: 14197.199 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Musca domestica (house fly)
#4: DNA chain DNA (8-MER)


Mass: 2162.448 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Musca domestica (house fly)

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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: Two right-end Hermes transpososome / Type: COMPLEX
Details: Hermes transposase tetramer of dimers complex bound to two transposon right-end DNAs. The complex was obtained by mixing the purified protein and the DNA.
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.627 MDa / Experimental value: NO
Source (natural)Organism: Musca domestica (house fly)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Top10 / Plasmid: pBAD/Myc-His
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.3 mMTCEPC9H15O6P1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The complex was formed in vitro by mixing the purified protein with the DNA and further purified by size exclusion chromatography.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.66 sec. / Electron dose: 48.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9500

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEMimage acquisition
4Gctf1.06CTF correction
5RELION3.1CTF correction
8UCSF Chimera1.13.1model fitting
9Coot0.9.6.2-pre ELmodel fitting
11PHENIX1.19.2model refinement
12RELION3.1initial Euler assignment
13RELION3.1final Euler assignment
14RELION3.1classification
15RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2920000
3D reconstructionResolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53656
Details: The 3D reconstruction is not representative of the complex in solution. The Hermes tranposase forms a tetramer of dimers assembled in a ring like shape. Only the Hermes dimers interacting ...Details: The 3D reconstruction is not representative of the complex in solution. The Hermes tranposase forms a tetramer of dimers assembled in a ring like shape. Only the Hermes dimers interacting with the DNAs have been partially reconstructed. Their BED domains did not show any density.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16DX0

6dx0

16DX01PDBexperimental model
24D1Q

4d1q

14D1Q2PDBexperimental model
38EB5

8eb5

18EB53PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00420605
ELECTRON MICROSCOPYf_angle_d0.63628751
ELECTRON MICROSCOPYf_dihedral_angle_d26.5214166
ELECTRON MICROSCOPYf_chiral_restr0.043269
ELECTRON MICROSCOPYf_plane_restr0.0042869

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