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- EMDB-33013: CryoEM structure of RuvA-Holliday junction complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33013
TitleCryoEM structure of RuvA-Holliday junction complex
Map data
Sample
  • Complex: RuvA-Holliday junction complex
    • Complex: Holliday junction
      • DNA: DNA (26-MER)
      • DNA: DNA (26-MER)
      • DNA: DNA (26-MER)
      • DNA: DNA (26-MER)
    • Complex: RuvA
      • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
KeywordsRuvA / Holliday junction / Homologous recombination / DNA damage repair / DNA BINDING PROTEIN-DNA COMPLEX
Function / homology
Function and homology information


Holliday junction helicase complex / Holliday junction resolvase complex / four-way junction helicase activity / four-way junction DNA binding / DNA recombination / DNA repair / ATP binding / cytoplasm
Similarity search - Function
Holliday junction DNA helicase RuvA, C-terminal / DNA helicase, Holliday junction RuvA type, domain I, bacterial / RuvA, C-terminal domain superfamily / RuvA N terminal domain / RuvA, C-terminal domain / Bacterial DNA recombination protein RuvA / RuvA domain 2-like / Helix-hairpin-helix domain / Helix-hairpin-helix DNA-binding motif, class 1 / Helix-hairpin-helix DNA-binding motif class 1 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Holliday junction branch migration complex subunit RuvA
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 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 4, 2022-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33013.png

Downloads & links

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Map

FileDownload / File: emd_33013.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.9260654 - 4.2261167
Average (Standard dev.)-0.000116294235 (±0.07806878)
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_33013_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_33013_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 : RuvA-Holliday junction complex

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

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Supramolecule #1: RuvA-Holliday junction complex

SupramoleculeName: RuvA-Holliday junction complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Holliday junction

SupramoleculeName: Holliday junction / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4

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Supramolecule #3: RuvA

SupramoleculeName: RuvA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Macromolecule #1: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.999266 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DT)(DA)(DT)(DA)(DA)(DT) (DA)(DT)(DT)(DT)(DA)(DA)(DT)(DA)(DT)(DT) (DA)(DT)(DA)(DA)(DT)(DA)

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Macromolecule #2: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 2 / 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 #3: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.981238 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DT)(DT)(DA)(DA)(DT)(DA) (DT)(DT)(DA)(DA)(DA)(DT)(DA)(DT)(DT)(DA) (DT)(DA)(DT)(DT)(DT)(DT)

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Macromolecule #4: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 4 / 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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Macromolecule #5: Holliday junction ATP-dependent DNA helicase RuvA

MacromoleculeName: Holliday junction ATP-dependent DNA helicase RuvA / type: protein_or_peptide / ID: 5 / Number of copies: 8 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1
Molecular weightTheoretical: 15.432009 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MIGRLRGTLA EKQPPHLILD VNGVGYEVEV PMTTLYRLPS VGEPVTLHTH LVVREDAHLL YGFAEKRERE LFRELIRLNG VGPKLALAL MSGLEVDELV RCVQAQDTST LVKIPGVGKK TAERLLVELK DRFKAWEN

UniProtKB: Holliday junction branch migration complex subunit RuvA

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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: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 143397
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-7x5a:
CryoEM structure of RuvA-Holliday junction complex

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