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- EMDB-28101: Structure of single homo-hexameric Holliday junction ATP-dependen... -

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Basic information

Entry
Database: EMDB / ID: EMD-28101
TitleStructure of single homo-hexameric Holliday junction ATP-dependent DNA helicase RuvB motor
Map data
Sample
  • Complex: Single homo-hexameric AAA+ ATPase RuvB motor
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvB
    • DNA: 49-mer DNA
    • DNA: 51-mer DNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsHolliday Junction / AAA+ ATPase / RuvB / Homo-hexamer / DNA recombination / STRUCTURAL PROTEIN
Function / homology
Function and homology information


Holliday junction resolvase complex / four-way junction helicase activity / four-way junction DNA binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA recombination / DNA repair / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
DNA helicase, Holliday junction RuvB-type / RuvB C-terminal winged helix domain / RuvB-like P-loop domain / RuvB, AAA lid domain / RuvB C-terminal winged helix domain / Holliday junction DNA helicase RuvB P-loop domain / RuvB AAA lid domain / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities ...DNA helicase, Holliday junction RuvB-type / RuvB C-terminal winged helix domain / RuvB-like P-loop domain / RuvB, AAA lid domain / RuvB C-terminal winged helix domain / Holliday junction DNA helicase RuvB P-loop domain / RuvB AAA lid domain / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Holliday junction branch migration complex subunit RuvB
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsShen ZF / Rish AD / Fu TM
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: Molecular mechanisms of Holliday junction branch migration catalyzed by an asymmetric RuvB hexamer.
Authors: Anthony D Rish / Zhangfei Shen / Zhenhang Chen / Nan Zhang / Qingfei Zheng / Tian-Min Fu /
Abstract: The Holliday junction (HJ) is a DNA intermediate of homologous recombination, involved in many fundamental physiological processes. RuvB, an ATPase motor protein, drives branch migration of the ...The Holliday junction (HJ) is a DNA intermediate of homologous recombination, involved in many fundamental physiological processes. RuvB, an ATPase motor protein, drives branch migration of the Holliday junction with a mechanism that had yet to be elucidated. Here we report two cryo-EM structures of RuvB, providing a comprehensive understanding of HJ branch migration. RuvB assembles into a spiral staircase, ring-like hexamer, encircling dsDNA. Four protomers of RuvB contact the DNA backbone with a translocation step size of 2 nucleotides. The variation of nucleotide-binding states in RuvB supports a sequential model for ATP hydrolysis and nucleotide recycling, which occur at separate, singular positions. RuvB's asymmetric assembly also explains the 6:4 stoichiometry between the RuvB/RuvA complex, which coordinates HJ migration in bacteria. Taken together, we provide a mechanistic understanding of HJ branch migration facilitated by RuvB, which may be universally shared by prokaryotic and eukaryotic organisms.
History
DepositionSep 9, 2022-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28101.png

Downloads & links

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Map

FileDownload / File: emd_28101.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 216 pix.
= 233.28 Å		1.08 Å/pix.
x 216 pix.
= 233.28 Å		1.08 Å/pix.
x 216 pix.
= 233.28 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.314
Minimum - Maximum-2.4995036 - 3.927434
Average (Standard dev.)0.0030204488 (±0.09500741)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 233.28001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28101_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28101_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Single homo-hexameric AAA+ ATPase RuvB motor

EntireName: Single homo-hexameric AAA+ ATPase RuvB motor
Components
  • Complex: Single homo-hexameric AAA+ ATPase RuvB motor
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvB
    • DNA: 49-mer DNA
    • DNA: 51-mer DNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Single homo-hexameric AAA+ ATPase RuvB motor

SupramoleculeName: Single homo-hexameric AAA+ ATPase RuvB motor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Macromolecule #1: Holliday junction ATP-dependent DNA helicase RuvB

MacromoleculeName: Holliday junction ATP-dependent DNA helicase RuvB / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: ATCC 27634 / DSM 579 / HB8
Molecular weightTheoretical: 36.024688 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEDLALRPKT LDEYIGQERL KQKLRVYLEA AKARKEPLEH LLLFGPPGLG KTTLAHVIAH ELGVNLRVTS GPAIEKPGDL AAILANSLE EGDILFIDEI HRLSRQAEEH LYPAMEDFVM DIVIGQGPAA RTIRLELPRF TLIGATTRPG LITAPLLSRF G IVEHLEYY ...String:
MEDLALRPKT LDEYIGQERL KQKLRVYLEA AKARKEPLEH LLLFGPPGLG KTTLAHVIAH ELGVNLRVTS GPAIEKPGDL AAILANSLE EGDILFIDEI HRLSRQAEEH LYPAMEDFVM DIVIGQGPAA RTIRLELPRF TLIGATTRPG LITAPLLSRF G IVEHLEYY TPEELAQGVM RDARLLGVRI TEEAALEIGR RSRGTMRVAK RLFRRVRDFA QVAGEEVITR ERALEALAAL GL DELGLEK RDREILEVLI LRFGGGPVGL ATLATALSED PGTLEEVHEP YLIRQGLLKR TPRGRVATEL AYRHLGYPPP VGP LLEP

UniProtKB: Holliday junction branch migration complex subunit RuvB

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Macromolecule #2: 49-mer DNA

MacromoleculeName: 49-mer DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 15.146732 KDa
SequenceString:
(DA)(DG)(DA)(DA)(DT)(DC)(DT)(DG)(DC)(DC) (DG)(DA)(DG)(DA)(DG)(DA)(DC)(DC)(DG)(DA) (DG)(DC)(DA)(DG)(DA)(DA)(DT)(DT)(DC) (DT)(DA)(DT)(DG)(DT)(DG)(DT)(DT)(DT)(DA) (DC) (DC)(DA)(DA)(DG)(DC)(DG)(DC)(DT) (DG)

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Macromolecule #3: 51-mer DNA

MacromoleculeName: 51-mer DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 15.69107 KDa
SequenceString:
(DC)(DA)(DG)(DC)(DG)(DC)(DT)(DT)(DG)(DG) (DT)(DA)(DA)(DA)(DC)(DA)(DC)(DA)(DT)(DA) (DG)(DA)(DA)(DT)(DT)(DC)(DT)(DG)(DC) (DT)(DC)(DT)(DC)(DG)(DG)(DT)(DC)(DT)(DG) (DA) (DG)(DC)(DC)(DG)(DT)(DC)(DT)(DA) (DA)(DG)(DA)

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 3 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1ixs

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 695592
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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