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- PDB-8edg: Cryo-EM structure of the Hermes transposase bound to two left-end... -

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Basic information

Entry
Database: PDB / ID: 8edg
TitleCryo-EM structure of the Hermes transposase bound to two left-ends of its DNA transposon
Components
  • DNA (46-MER)
  • DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3')
  • DNA (55-MER)
  • Hermes transposase
KeywordsRECOMBINATION/DNA / transposase / transpososome / BED domain / protein-DNA complex / RECOMBINATION / RECOMBINATION-DNA complex
Function / homology
Function and homology information


nucleic acid metabolic process / protein dimerization activity / DNA binding / metal ion binding / nucleus
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: 4.64 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
DepositionSep 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
M: DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3')
N: DNA (46-MER)
O: DNA (55-MER)
Q: DNA (46-MER)
R: DNA (55-MER)
P: DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3')
A: Hermes transposase
C: Hermes transposase
I: Hermes transposase
E: Hermes transposase
G: Hermes transposase
K: Hermes transposase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)488,19118
Polymers487,79812
Non-polymers3926
Water0
1


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

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Components

#1: DNA chain DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3')


Mass: 2162.448 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Musca domestica (house fly)
#2: DNA chain DNA (46-MER)


Mass: 14173.139 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Musca domestica (house fly)
#3: DNA chain DNA (55-MER)


Mass: 16931.867 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Musca domestica (house fly)
#4: Protein
Hermes transposase


Mass: 70210.570 Da / Num. of mol.: 6 / Mutation: Q2E,K128G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Musca domestica (house fly) / Plasmid: pBAD/Myc-His / Production host: Escherichia coli (E. coli) / Strain (production host): Top10 / References: UniProt: Q25438
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestY

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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 left-end Hermes transpososome / Type: COMPLEX
Details: Hermes transposase tetramer of dimers complex bound to two transposon left-end DNAs. The complex was obtained by mixing the purified protein and the DNA.
Entity ID: #1-#4 / 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)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
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 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

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 22.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / 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
4RELION3.1CTF correction
7UCSF Chimera1.13.1model fitting
8Coot0.9model fitting
10PHENIX1.19.2model refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3850000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143400
Details: The 3D reconstruction is not representative of the complex in solution. The Hermes transposase forms a tetramer of dimers assembled in a ring like shape. Due to their low resolution two ...Details: The 3D reconstruction is not representative of the complex in solution. The Hermes transposase forms a tetramer of dimers assembled in a ring like shape. Due to their low resolution two Hermes dimers have been partially or completely masked out during the processing. Only the opposite two Hermes dimers interacting with the DNAs have been fully reconstructed, along with the two DNAs and the two BED domains of a third Hermes dimer.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
16DX0

6dx0

1
24D1Q

4d1q

1
38EB5

8eb5

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 173.07 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00324244
ELECTRON MICROSCOPYf_angle_d0.57433640
ELECTRON MICROSCOPYf_dihedral_angle_d24.8044640
ELECTRON MICROSCOPYf_chiral_restr0.0383818
ELECTRON MICROSCOPYf_plane_restr0.0043470

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