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- EMDB-33044: CryoEM structure of RuvA-RuvB-Holliday junction complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33044
TitleCryoEM structure of RuvA-RuvB-Holliday junction complex
Map data
Sample
  • Complex: RuvA-RuvB-Holliday junction complex
    • Complex: RuvA-RuvB
      • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
      • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvB
    • Complex: DNA
      • DNA: DNA (40-MER)
      • DNA: DNA (40-MER)
      • DNA: DNA (26-MER)
      • DNA: DNA (26-MER)
KeywordsHolliday junction / Homologous recombination / DNA damage repair / Branch migration / RECOMBINATION-DNA COMPLEX
Function / homology
Function and homology information


cellular response to chromate / Holliday junction helicase complex / cellular response to selenite ion / Holliday junction resolvase complex / SOS response / four-way junction DNA binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA recombination / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity ...cellular response to chromate / Holliday junction helicase complex / cellular response to selenite ion / Holliday junction resolvase complex / SOS response / four-way junction DNA binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA recombination / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / DNA repair / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Bacterial DNA recombination protein RuvA / DNA helicase, Holliday junction RuvB-type / RuvB C-terminal winged helix domain / RuvB-like P-loop domain / Holliday junction DNA helicase RuvA, C-terminal / DNA helicase, Holliday junction RuvA type, domain I, bacterial / RuvA, C-terminal domain superfamily / RuvB, AAA lid domain / RuvA N terminal domain / RuvB C-terminal winged helix domain ...Bacterial DNA recombination protein RuvA / DNA helicase, Holliday junction RuvB-type / RuvB C-terminal winged helix domain / RuvB-like P-loop domain / Holliday junction DNA helicase RuvA, C-terminal / DNA helicase, Holliday junction RuvA type, domain I, bacterial / RuvA, C-terminal domain superfamily / RuvB, AAA lid domain / RuvA N terminal domain / RuvB C-terminal winged helix domain / Holliday junction DNA helicase RuvB P-loop domain / RuvA, C-terminal domain / RuvB AAA lid domain / RuvA domain 2-like / Helix-hairpin-helix domain / Helix-hairpin-helix DNA-binding motif, class 1 / Helix-hairpin-helix DNA-binding motif class 1 / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / 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 RuvA / Holliday junction branch migration complex subunit RuvB
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.02 Å
AuthorsLin Z / Qu Q / Zhang X / Zhou Z
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971222 China
National Natural Science Foundation of China (NSFC)32171194 China
CitationJournal: Front Plant Sci / Year: 2023
Title: Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from .
Authors: Xu Zhang / Zixuan Zhou / Lin Dai / Yulin Chao / Zheng Liu / Mingdong Huang / Qianhui Qu / Zhonghui Lin /
Abstract: Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB ...Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. (, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from . The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal β-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA.
History
DepositionMar 10, 2022-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33044.png

Downloads & links

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Map

FileDownload / File: emd_33044.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 398.4 Å		0.83 Å/pix.
x 480 pix.
= 398.4 Å		0.83 Å/pix.
x 480 pix.
= 398.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.6472173 - 1.1958688
Average (Standard dev.)-0.00004795316 (±0.028333388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 398.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33044_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_33044_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : RuvA-RuvB-Holliday junction complex

EntireName: RuvA-RuvB-Holliday junction complex
Components
  • Complex: RuvA-RuvB-Holliday junction complex
    • Complex: RuvA-RuvB
      • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
      • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvB
    • Complex: DNA
      • DNA: DNA (40-MER)
      • DNA: DNA (40-MER)
      • DNA: DNA (26-MER)
      • DNA: DNA (26-MER)

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Supramolecule #1: RuvA-RuvB-Holliday junction complex

SupramoleculeName: RuvA-RuvB-Holliday junction complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #2: RuvA-RuvB

SupramoleculeName: RuvA-RuvB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: synthetic construct (others)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#6

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

MacromoleculeName: Holliday junction ATP-dependent DNA helicase RuvA / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1
Molecular weightTheoretical: 21.987594 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIGRLRGTLA EKQPPHLILD VNGVGYEVEV PMTTLYRLPS VGEPVTLHTH LVVREDAHLL YGFAEKRERE LFRELIRLNG VGPKLALAL MSGLEVDELV RCVQAQDTST LVKIPGVGKK TAERLLVELK DRFKAWENMP TIAPLVMEPR ASATVSSAEA D AVSALIAL ...String:
MIGRLRGTLA EKQPPHLILD VNGVGYEVEV PMTTLYRLPS VGEPVTLHTH LVVREDAHLL YGFAEKRERE LFRELIRLNG VGPKLALAL MSGLEVDELV RCVQAQDTST LVKIPGVGKK TAERLLVELK DRFKAWENMP TIAPLVMEPR ASATVSSAEA D AVSALIAL GFKPQEASRA VAAVPGEDLS SEEMIRQALK GMV

UniProtKB: Holliday junction branch migration complex subunit RuvA

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Macromolecule #2: Holliday junction ATP-dependent DNA helicase RuvB

MacromoleculeName: Holliday junction ATP-dependent DNA helicase RuvB / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1
Molecular weightTheoretical: 38.981578 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIEPDRLISA VSGRERDEQL DRAIRPLKLA DYIGQPSVRE QMELFIHAAR GRQEALDHTL IFGPPGLGKT TLANIIAQEM GVSIKSTSG PVLERPGDLA ALLTNLEAGD VLFVDEIHRL SPIVEEVLYP AMEDFQLDIM IGEGPAARSI KLDLPPFTLV G ATTRAGML ...String:
MIEPDRLISA VSGRERDEQL DRAIRPLKLA DYIGQPSVRE QMELFIHAAR GRQEALDHTL IFGPPGLGKT TLANIIAQEM GVSIKSTSG PVLERPGDLA ALLTNLEAGD VLFVDEIHRL SPIVEEVLYP AMEDFQLDIM IGEGPAARSI KLDLPPFTLV G ATTRAGML TNPLRDRFGI VQRLEFYNVE DLATIVSRSA GILGLEIEPQ GAAEIAKRAR GTPRIANRLL RRVRDFAEVR GQ GDITRVI ADKALNLLDV DERGFDHLDR RLLLTMIDKF DGGPVGIDNL AAALSEERHT IEDVLEPYLI QQGYIMRTPR GRV VTRHAY LHFGLNIPKR LGPGVTTDLF TSEDGN

UniProtKB: Holliday junction branch migration complex subunit RuvB

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Macromolecule #3: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.321062 KDa
SequenceString:
(DA)(DT)(DA)(DT)(DT)(DA)(DT)(DA)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DA)(DT)(DA)(DA)(DT) (DA)(DA)(DA)(DT)(DA)(DT)(DT)(DT)(DA) (DA)(DT)(DA)(DT)(DT)(DA)(DT)(DA)(DA)(DT) (DA)

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Macromolecule #4: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.303033 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DT)(DT)(DA)(DA)(DT)(DA) (DT)(DT)(DA)(DA)(DA)(DT)(DA)(DT)(DT)(DT) (DA)(DT)(DT)(DA)(DT)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DT)(DA)(DT)(DA)(DA)(DT)(DA) (DT)

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Macromolecule #5: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.972225 KDa
SequenceString:
(DT)(DA)(DT)(DT)(DA)(DT)(DA)(DA)(DT)(DA) (DT)(DT)(DA)(DA)(DA)(DT)(DA)(DT)(DT)(DA) (DT)(DA)(DT)(DT)(DT)(DA)

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Macromolecule #6: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.972224 KDa
SequenceString:
(DT)(DA)(DA)(DA)(DT)(DA)(DT)(DA)(DA)(DT) (DA)(DT)(DT)(DT)(DA)(DA)(DT)(DA)(DT)(DT) (DA)(DA)(DT)(DT)(DT)(DT)

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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: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 260.0 kPa
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 60241 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 20536
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7x7q:
CryoEM structure of RuvA-RuvB-Holliday junction complex

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