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- EMDB-15085: 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 - composite map 1 and 2 | |||||||||
![]() | RuvAB branch migration motor complexed to the Holliday junction - composite map 2 [dataset t2] | |||||||||
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![]() | DNA recombination / DNA repair / branch migration / Holliday junction / helicase / DNA BINDING PROTEIN | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.0 Å | |||||||||
![]() | Wald J / Marlovits TC | |||||||||
Funding support | ![]()
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![]() | ![]() 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. | |||||||||
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 4.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.2 KB 14.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 12.9 KB | Display | ![]() |
Images | ![]() | 92.4 KB | ||
Filedesc metadata | ![]() | 4.4 KB | ||
Others | ![]() | 10.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7pblC ![]() 7pbmC ![]() 7pbnC ![]() 7pboC ![]() 7pbpC ![]() 7pbqC ![]() 7pbrC ![]() 7pbsC ![]() 7pbtC ![]() 7pbuC C: citing same article ( |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | RuvAB branch migration motor complexed to the Holliday junction - composite map 2 [dataset t2] | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.09 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: RuvAB branch migration motor complexed to the Holliday...
File | emd_15085_additional_1.map | ||||||||||||
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Annotation | RuvAB branch migration motor complexed to the Holliday junction - composite map 1 [dataset t2] | ||||||||||||
Projections & Slices |
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Density Histograms |
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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 - composite maps [t2] |
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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 - composite maps [t2] type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: RuvAB branch migration complex |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 650 KDa |
-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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR |
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 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 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 |