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- EMDB-28034: Cryo-EM structure of the Hermes transposase bound to two left-end... -

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Basic information

Entry
Database: EMDB / ID: EMD-28034
TitleCryo-EM structure of the Hermes transposase bound to two left-ends of its DNA transposon
Map data
Sample
  • Complex: Two left-end Hermes transpososome
    • DNA: DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3')
    • DNA: DNA (46-MER)
    • DNA: DNA (55-MER)
    • Protein or peptide: Hermes transposase
  • Ligand: ZINC ION
Keywordstransposase / 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 / 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
Biological speciesMusca domestica (house fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.64 Å
AuthorsLannes L / Dyda F
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28034.png

Downloads & links

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Map

FileDownload / File: emd_28034.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.0104
Minimum - Maximum-0.031263 - 0.06668972
Average (Standard dev.)0.000080967446 (±0.002763421)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 306.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28034_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28034_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Two left-end Hermes transpososome

EntireName: Two left-end Hermes transpososome
Components
  • Complex: Two left-end Hermes transpososome
    • DNA: DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3')
    • DNA: DNA (46-MER)
    • DNA: DNA (55-MER)
    • Protein or peptide: Hermes transposase
  • Ligand: ZINC ION

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Supramolecule #1: Two left-end Hermes transpososome

SupramoleculeName: Two left-end Hermes transpososome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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.
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 627 KDa

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

MacromoleculeName: DNA (5'-D(*GP*CP*GP*TP*GP*AP*A)-3') / type: dna / ID: 1 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 2.162448 KDa
SequenceString:
(DG)(DC)(DG)(DT)(DG)(DA)(DA)

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Macromolecule #2: DNA (46-MER)

MacromoleculeName: DNA (46-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 14.173139 KDa
SequenceString:
(DA)(DG)(DA)(DG)(DA)(DA)(DC)(DA)(DA)(DC) (DA)(DA)(DC)(DA)(DA)(DG)(DT)(DG)(DG)(DC) (DT)(DT)(DA)(DT)(DT)(DT)(DT)(DG)(DA) (DT)(DA)(DC)(DT)(DT)(DA)(DT)(DG)(DC)(DG) (DC) (DC)(DA)(DC)(DT)(DT)(DG)

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Macromolecule #3: DNA (55-MER)

MacromoleculeName: DNA (55-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 16.931867 KDa
SequenceString: (DC)(DA)(DA)(DG)(DT)(DG)(DG)(DC)(DG)(DC) (DA)(DT)(DA)(DA)(DG)(DT)(DA)(DT)(DC)(DA) (DA)(DA)(DA)(DT)(DA)(DA)(DG)(DC)(DC) (DA)(DC)(DT)(DT)(DG)(DT)(DT)(DG)(DT)(DT) (DG) (DT)(DT)(DC)(DT)(DC)(DT) ...String:
(DC)(DA)(DA)(DG)(DT)(DG)(DG)(DC)(DG)(DC) (DA)(DT)(DA)(DA)(DG)(DT)(DA)(DT)(DC)(DA) (DA)(DA)(DA)(DT)(DA)(DA)(DG)(DC)(DC) (DA)(DC)(DT)(DT)(DG)(DT)(DT)(DG)(DT)(DT) (DG) (DT)(DT)(DC)(DT)(DC)(DT)(DG)(DG) (DT)(DT)(DC)(DA)(DC)(DG)(DC)

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Macromolecule #4: Hermes transposase

MacromoleculeName: Hermes transposase / type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 70.21057 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEKMDNLEVK AKINQGLYKI TPRHKGTSFI WNVLADIQKE DDTLVEGWVF CRKCEKVLKY TTRQTSNLCR HKCCASLKQS RELKTVSAD CKKEAIEKCA QWVVRDCRPF SAVSGSGFID MIKFFIKVGA EYGEHVNVEE LLPSPITLSR KVTSDAKEKK A LISREIKS ...String:
MEKMDNLEVK AKINQGLYKI TPRHKGTSFI WNVLADIQKE DDTLVEGWVF CRKCEKVLKY TTRQTSNLCR HKCCASLKQS RELKTVSAD CKKEAIEKCA QWVVRDCRPF SAVSGSGFID MIKFFIKVGA EYGEHVNVEE LLPSPITLSR KVTSDAKEKK A LISREIKS AVEKDGASAT IDLWTDNYIK RNFLGVTLHY HENNELRDLI LGLKSLDFER STAENIYKKL KAIFSQFNVE DL SSIKFVT DRGANVVKSL ANNIRINCSS HLLSNVLENS FEETPELNMP ILACKNIVKY FKKANLQHRL RSSLKSECPT RWN STYTML RSILDNWESV IQILSEAGET QRIVHINKSI IQTMVNILDG FERIFKELQT CSSPSLCFVV PSILKVKEIC SPDV GDVAD IAKLKVNIIK NVRIIWEENL SIWHYTAFFF YPPALHMQQE KVAQIKEFCL SKMEDLELIN RMSSFNELSA TQLNQ SDSN SHNSIDLTSH SKDISTTSFF FPQLTQNNSR EPPVCPSDEF EFYRKEIVIL SEDFKVMEWW NLNSKKYPKL SKLALS LLS IPASSAASER TFSLAGNIIT EKRNRIGQQT VDSLLFLNSF YKNFCKLDI

UniProtKB: Hermes transposase

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 6 / 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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
0.3 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsThe complex was formed in vitro by mixing the purified protein with the DNA and further purified by size exclusion chromatography.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 9500 / Average exposure time: 2.0 sec. / Average electron dose: 22.3 e/Å2

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Image processing

Particle selectionNumber selected: 3850000
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
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.
Number images used: 143400
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6dx0

source_name: PDB, initial_model_type: experimental model

4d1q

source_name: PDB, initial_model_type: experimental model

8eb5

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8edg:
Cryo-EM structure of the Hermes transposase bound to two left-ends of its DNA transposon

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