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- EMDB-13294: RuvAB branch migration motor complexed to the Holliday junction -... -

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Basic information

Entry
Database: EMDB / ID: EMD-13294
TitleRuvAB branch migration motor complexed to the Holliday junction - RuvB AAA+ state s1 [t2 dataset]
Map dataRuvB AAA hexamer state s1 (t2)
Sample
  • Complex: RuvAB branch migration motor complexed to the Holliday junction - RuvB motor state s1 [t2 dataset]
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvB
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
    • DNA: random DNA sequence
    • DNA: random DNA sequence
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
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 helicase / DNA repair / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
DNA helicase, Holliday junction RuvB-type / RuvB C-terminal winged helix 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 / RuvA, C-terminal domain / RuvB AAA lid domain ...DNA helicase, Holliday junction RuvB-type / RuvB C-terminal winged helix 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 / RuvA, C-terminal domain / RuvB AAA lid domain / Bacterial DNA recombination protein RuvA / RuvB-like P-loop domain / Holliday junction DNA helicase RuvB P-loop 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / 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 speciesStreptococcus thermophilus (bacteria) / Salmonella typhimurium (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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-
UpdateNov 30, 2022-
Current statusNov 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13294.png

Downloads & links

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Map

FileDownload / File: emd_13294.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRuvB AAA hexamer state s1 (t2)
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.058565255 - 0.13769865
Average (Standard dev.)6.2162646e-05 (±0.0016833046)
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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Half map: RuvB AAA hexamer state s1 (t2)

Fileemd_13294_half_map_1.map
AnnotationRuvB AAA hexamer state s1 (t2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RuvB AAA hexamer state s1 (t2)

Fileemd_13294_half_map_2.map
AnnotationRuvB AAA hexamer state s1 (t2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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 - RuvB motor state s1 [t2 dataset]
Components
  • Complex: RuvAB branch migration motor complexed to the Holliday junction - RuvB motor state s1 [t2 dataset]
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvB
    • Protein or peptide: Holliday junction ATP-dependent DNA helicase RuvA
    • DNA: random DNA sequence
    • DNA: random DNA sequence
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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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 - RuvB motor state s1 [t2 dataset]
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: RuvB helicase
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Recombinant plasmid: pProEX HTB
Molecular weightTheoretical: 220 KDa

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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: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 35.447508 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: TLRPQYFKEY IGQDKVKDQL KIFIEAAKLR DEALDHTLLF GPPGLGKTTM AFVIANEMGV NLKQTSGPAI EKAGDLVAIL NDLEPGDIL FIDEIHRMPM AVEEVLYSAM EDYYIDIMIG AGETSRSVHL DLPPFTLVGA TTRAGMLSNP LRARFGINGH M EYYELPDL ...String:
TLRPQYFKEY IGQDKVKDQL KIFIEAAKLR DEALDHTLLF GPPGLGKTTM AFVIANEMGV NLKQTSGPAI EKAGDLVAIL NDLEPGDIL FIDEIHRMPM AVEEVLYSAM EDYYIDIMIG AGETSRSVHL DLPPFTLVGA TTRAGMLSNP LRARFGINGH M EYYELPDL TEIVERTSEI FEMTITPEAA LELARRSRGT PRIANRLLKR VRDYAQIMGD GVIDDKIADQ ALTMLDVDHE GL DYVDQKI LRTMIEMYGG GPVGLGTLSV NIAEERETVE DMYEPYLIQK GFIMRTRTGR VATAKAYEHM GYDYTRDN

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

MacromoleculeName: Holliday junction ATP-dependent DNA helicase RuvA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Salmonella typhimurium (bacteria)
Molecular weightTheoretical: 5.105734 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
SEDAEQEAVA ALVALGYKPQ EASRMVSKIA RPDASSETLI RDALRAAL

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Macromolecule #3: random DNA sequence

MacromoleculeName: random DNA sequence / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.643037 KDa
SequenceString:
(DG)(DA)(DA)(DC)(DC)(DT)(DT)(DC)(DG)(DA) (DG)(DG)(DA)(DA)(DG)

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Macromolecule #4: random DNA sequence

MacromoleculeName: random DNA sequence / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.535946 KDa
SequenceString:
(DC)(DT)(DT)(DC)(DC)(DT)(DC)(DG)(DA)(DA) (DG)(DG)(DT)(DT)(DC)

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / 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 #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / 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: 8
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.15000000000000002 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
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

CTF correctionSoftware - Name: CTFFIND
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 102619
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-7pbl:
RuvAB branch migration motor complexed to the Holliday junction - RuvB AAA+ state s1 [t2 dataset]

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