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- EMDB-13301: RuvAB branch migration motor complexed to the Holliday junction -... -
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Open data
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Basic information
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Title | RuvAB branch migration motor complexed to the Holliday junction - RuvB AAA+ state s0+A [t1 dataset] | |||||||||
![]() | RuvB AAA state s0 A [t1 dataset] | |||||||||
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![]() | DNA recombination / DNA repair / branch migration / Holliday junction / helicase / HYDROLASE | |||||||||
Function / homology | ![]() Holliday junction helicase complex / Holliday junction resolvase complex / four-way junction helicase activity / four-way junction DNA binding / DNA helicase / DNA recombination / DNA repair / ATP hydrolysis activity / ATP binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
![]() | Wald J / Marlovits TC | |||||||||
![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 12.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.7 KB 15.7 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 12.8 KB | Display | ![]() |
Images | ![]() | 94 KB | ||
Filedesc metadata | ![]() | 11.5 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7pbsMC ![]() 7pblC ![]() 7pbmC ![]() 7pbnC ![]() 7pboC ![]() 7pbpC ![]() 7pbqC ![]() 7pbrC ![]() 7pbtC ![]() 7pbuC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | RuvB AAA state s0 A [t1 dataset] | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : RuvAB branch migration motor complexed to the Holliday junction -...
Entire | Name: RuvAB branch migration motor complexed to the Holliday junction - RuvB AAA+ state s0+A [t1 dataset] |
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Components |
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-Supramolecule #1: RuvAB branch migration motor complexed to the Holliday junction -...
Supramolecule | Name: RuvAB branch migration motor complexed to the Holliday junction - RuvB AAA+ state s0+A [t1 dataset] type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: RuvB helicase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 220 KDa |
-Macromolecule #1: Holliday junction ATP-dependent DNA helicase RuvB
Macromolecule | Name: Holliday junction ATP-dependent DNA helicase RuvB / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 35.447508 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 UniProtKB: Holliday junction branch migration complex subunit RuvB |
-Macromolecule #2: Holliday junction ATP-dependent DNA helicase RuvA
Macromolecule | Name: Holliday junction ATP-dependent DNA helicase RuvA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 5.105734 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: SEDAEQEAVA ALVALGYKPQ EASRMVSKIA RPDASSETLI RDALRAAL UniProtKB: Holliday junction branch migration complex subunit RuvA |
-Macromolecule #3: random DNA
Macromolecule | Name: random DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.643037 KDa |
Sequence | String: (DG)(DA)(DA)(DC)(DC)(DT)(DT)(DC)(DG)(DA) (DG)(DG)(DA)(DA)(DG) |
-Macromolecule #4: random DNA
Macromolecule | Name: random DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.535946 KDa |
Sequence | String: (DC)(DT)(DT)(DC)(DC)(DT)(DC)(DG)(DA)(DA) (DG)(DG)(DT)(DT)(DC) |
-Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 5 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ![]() ChemComp-AGS: |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #7: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ![]() ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 8 |
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Grid | Model: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.15 |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
Details | in-vitro reconstituted freshly before vitrification |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number real images: 10057 / Average exposure time: 3.0 sec. / Average electron dose: 53.3 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 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 stage | Specimen 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
-Atomic model buiding 1
Refinement | Protocol: OTHER |
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Output model | ![]() PDB-7pbs: |