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- EMDB-15085: RuvAB branch migration motor complexed to the Holliday junction -... -

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Basic information

Entry
Database: EMDB / ID: EMD-15085
TitleRuvAB branch migration motor complexed to the Holliday junction - composite map 1 and 2
Map dataRuvAB branch migration motor complexed to the Holliday junction - composite map 2 [dataset t2]
Sample
  • Complex: RuvAB branch migration motor complexed to the Holliday junction - composite maps [t2]
KeywordsDNA recombination / DNA repair / branch migration / Holliday junction / helicase / DNA BINDING PROTEIN
Biological speciesStreptococcus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsWald J / Marlovits TC
Funding support Germany, 1 items
OrganizationGrant numberCountry
Other government Germany
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
DepositionJun 1, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15085.png

Downloads & links

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Map

FileDownload / File: emd_15085.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRuvAB branch migration motor complexed to the Holliday junction - composite map 2 [dataset t2]
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: 0.01
Minimum - Maximum0.0 - 0.07793623
Average (Standard dev.)0.00015595941 (±0.0017156143)
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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Additional map: RuvAB branch migration motor complexed to the Holliday...

Fileemd_15085_additional_1.map
AnnotationRuvAB branch migration motor complexed to the Holliday junction - composite map 1 [dataset 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 - composite maps [t2]
Components
  • Complex: RuvAB branch migration motor complexed to the Holliday junction - composite maps [t2]

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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 - composite maps [t2]
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: RuvAB branch migration complex
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 650 KDa

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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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 529554
FSC plot (resolution estimation)

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