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- EMDB-13303: RuvAB branch migration motor complexed to the Holliday junction -... -

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Basic information

Entry
Database: EMDB / ID: EMD-13303
TitleRuvAB branch migration motor complexed to the Holliday junction - RuvA-HJ core [t2 dataset]
Map dataRuvA-HJ core [t2 dataset]
Sample
  • Complex: RuvAB branch migration motor complexed to the Holliday junction - RuvA-HJ core [t2 dataset]
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
    • DNA: Holliday junction
    • DNA: Holliday junction
KeywordsDNA recombination / DNA repair / branch migration / Holliday junction / helicase / HYDROLASE
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
Bacterial DNA recombination protein RuvA / 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 / 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 speciesStreptococcus thermophilus (bacteria) / Salmonella typhimurium (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWald J / Marlovits TC
CitationJournal: Nature / Year: 2022
Title: Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.
Authors: Jiri Wald / Dirk Fahrenkamp / Nikolaus Goessweiner-Mohr / Wolfgang Lugmayr / Luciano Ciccarelli / Oliver Vesper / Thomas C Marlovits /
Abstract: The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, ...The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.
History
DepositionAug 2, 2021-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13303.png

Downloads & links

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Map

FileDownload / File: emd_13303.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRuvA-HJ core [t2 dataset]
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 360 pix.
= 392.4 Å		1.09 Å/pix.
x 360 pix.
= 392.4 Å		1.09 Å/pix.
x 360 pix.
= 392.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.07224281 - 0.121615835
Average (Standard dev.)0.000008070986 (±0.0015752299)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 392.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : RuvAB branch migration motor complexed to the Holliday junction -...

EntireName: RuvAB branch migration motor complexed to the Holliday junction - RuvA-HJ core [t2 dataset]
Components
  • Complex: RuvAB branch migration motor complexed to the Holliday junction - RuvA-HJ core [t2 dataset]
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
    • DNA: Holliday junction
    • DNA: Holliday junction

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Supramolecule #1: RuvAB branch migration motor complexed to the Holliday junction -...

SupramoleculeName: RuvAB branch migration motor complexed to the Holliday junction - RuvA-HJ core [t2 dataset]
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: RuvA helicase
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 200 KDa

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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: Salmonella typhimurium (bacteria)
Molecular weightTheoretical: 14.860438 KDa
Recombinant expressionOrganism: Streptococcus thermophilus (bacteria)
SequenceString:
VIGRLRGIIL EKQPPIVLLE TGGVGYEVHM PMTCFYELPE AGQEAIVFTH FVVREDAQLL YGFNNKQERT LFKELIKTNG VGPKLALAI LSGMSAQQFV NAVEREELGA LVKLPGIGKK TAERLIVEMK DRFK

UniProtKB: Holliday junction branch migration complex subunit RuvA

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Macromolecule #2: Holliday junction

MacromoleculeName: Holliday junction / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.224434 KDa
SequenceString:
(DT)(DA)(DT)(DT)(DC)(DT)(DT)(DT)(DA)(DA) (DA)(DG)(DA)(DA)(DT)(DA)(DG)

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Macromolecule #3: Holliday junction

MacromoleculeName: Holliday junction / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.18441 KDa
SequenceString:
(DC)(DT)(DA)(DT)(DT)(DC)(DT)(DT)(DT)(DA) (DA)(DA)(DG)(DA)(DA)(DT)(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

BufferpH: 8
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.15
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsin-vitro reconstituted freshly before vitrification

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number real images: 30083 / Average exposure time: 5.0 sec. / Average electron dose: 30.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm
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 reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 229000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7pbu:
RuvAB branch migration motor complexed to the Holliday junction - RuvA-HJ core [t2 dataset]

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