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- PDB-7x5a: CryoEM structure of RuvA-Holliday junction complex -

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Basic information

Entry
Database: PDB / ID: 7x5a
TitleCryoEM structure of RuvA-Holliday junction complex
Components
  • (DNA (26-MER)) x 4
  • Holliday junction ATP-dependent DNA helicase RuvA
KeywordsDNA BINDING PROTEIN/DNA / RuvA / 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
DNA / DNA (> 10) / Holliday junction branch migration complex subunit RuvA
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsLin, Z. / Qu, Q. / Zhang, X. / Zhou, Z.
Funding support China, 2items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
I: DNA (26-MER)
J: DNA (26-MER)
K: DNA (26-MER)
L: DNA (26-MER)
A: Holliday junction ATP-dependent DNA helicase RuvA
B: Holliday junction ATP-dependent DNA helicase RuvA
C: Holliday junction ATP-dependent DNA helicase RuvA
D: Holliday junction ATP-dependent DNA helicase RuvA
E: Holliday junction ATP-dependent DNA helicase RuvA
F: Holliday junction ATP-dependent DNA helicase RuvA
G: Holliday junction ATP-dependent DNA helicase RuvA
H: Holliday junction ATP-dependent DNA helicase RuvA


Theoretical massNumber of molelcules
Total (without water)155,38112
Polymers155,38112
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain DNA (26-MER)


Mass: 7999.266 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (26-MER)


Mass: 7972.225 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (26-MER)


Mass: 7981.238 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (26-MER)


Mass: 7972.224 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Protein
Holliday junction ATP-dependent DNA helicase RuvA


Mass: 15432.009 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1 / Gene: ruvA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q51425, DNA helicase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1RuvA-Holliday junction complexCOMPLEXall0MULTIPLE SOURCES
2Holliday junctionCOMPLEX#1-#41SYNTHETIC
3RuvACOMPLEX#51RECOMBINANT
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 60241 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEM4image acquisition
4Gctf1.06CTF correction
5CTFFIND4.1.13CTF correction
12cryoSPARC3.1final Euler assignment
14cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143397 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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