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- EMDB-40553: Cryo-EM structure of the Hermes transposase bound to two right-en... -

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Basic information

Entry
Database: EMDB / ID: EMD-40553
TitleCryo-EM structure of the Hermes transposase bound to two right-ends of its DNA transposon.
Map data
Sample
  • Complex: Two right-end Hermes transpososome
    • Protein or peptide: Hermes transposase
    • DNA: DNA (55-MER)
    • DNA: DNA (46-MER)
    • DNA: DNA (8-MER)
Keywordstransposase / transpososome / BED domain / protein-DNA complex / 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
Biological speciesMusca domestica (house fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 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
DepositionApr 17, 2023-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40553.png

Downloads & links

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Map

FileDownload / File: emd_40553.map.gz / Format: CCP4 / Size: 93 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.01967928 - 0.044755302
Average (Standard dev.)0.00022606127 (±0.002215481)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions290290290
Spacing290290290
CellA=B=C: 249.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: half-maps processed with DeepEMhancer.

Fileemd_40553_additional_1.map
Annotationhalf-maps processed with DeepEMhancer.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40553_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_40553_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 right-end Hermes transpososome

EntireName: Two right-end Hermes transpososome
Components
  • Complex: Two right-end Hermes transpososome
    • Protein or peptide: Hermes transposase
    • DNA: DNA (55-MER)
    • DNA: DNA (46-MER)
    • DNA: DNA (8-MER)

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Supramolecule #1: Two right-end Hermes transpososome

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

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Macromolecule #1: Hermes transposase

MacromoleculeName: Hermes transposase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / 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 #2: DNA (55-MER)

MacromoleculeName: DNA (55-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 16.909779 KDa
SequenceString: (DC)(DT)(DT)(DA)(DT)(DC)(DT)(DA)(DT)(DG) (DT)(DG)(DG)(DC)(DT)(DT)(DA)(DC)(DG)(DT) (DT)(DT)(DG)(DC)(DC)(DT)(DG)(DT)(DG) (DG)(DC)(DT)(DT)(DG)(DT)(DT)(DG)(DA)(DA) (DG) (DT)(DT)(DC)(DT)(DC)(DT) ...String:
(DC)(DT)(DT)(DA)(DT)(DC)(DT)(DA)(DT)(DG) (DT)(DG)(DG)(DC)(DT)(DT)(DA)(DC)(DG)(DT) (DT)(DT)(DG)(DC)(DC)(DT)(DG)(DT)(DG) (DG)(DC)(DT)(DT)(DG)(DT)(DT)(DG)(DA)(DA) (DG) (DT)(DT)(DC)(DT)(DC)(DT)(DG)(DG) (DT)(DT)(DC)(DA)(DC)(DG)(DC)

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Macromolecule #3: DNA (46-MER)

MacromoleculeName: DNA (46-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Musca domestica (house fly)
Molecular weightTheoretical: 14.197199 KDa
SequenceString:
(DA)(DG)(DA)(DG)(DA)(DA)(DC)(DT)(DT)(DC) (DA)(DA)(DC)(DA)(DA)(DG)(DC)(DC)(DA)(DC) (DA)(DG)(DG)(DC)(DA)(DA)(DA)(DC)(DG) (DT)(DA)(DA)(DG)(DC)(DC)(DA)(DC)(DA)(DT) (DA) (DG)(DA)(DT)(DA)(DA)(DG)

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Macromolecule #4: DNA (8-MER)

MacromoleculeName: DNA (8-MER) / type: dna / ID: 4 / 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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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 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
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 KRIOS
Electron beamAcceleration voltage: 300 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: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9500 / Average exposure time: 1.66 sec. / Average electron dose: 48.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2920000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6dx0


Details: PDB ID 8EB5 and 4D1Q were also used.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.1 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 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.
Number images used: 53656
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-8sjd:
Cryo-EM structure of the Hermes transposase bound to two right-ends of its DNA transposon.

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