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- EMDB-36040: Composite map of the Rad51-ADP filament -

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Basic information

Entry
Database: EMDB / ID: EMD-36040
TitleComposite map of the Rad51-ADP filament
Map dataThis is a composite map with a high-resolution map (3.14 A) of the central part of the helix combined with the other part from a lower-resolution map (3.58A).
Sample
  • Complex: hRAD51-ADP filament intermediate
    • Protein or peptide: DNA repair protein RAD51 homolog 1
KeywordsRecombinase / ATPase / DNA repair protein / DNA binding / RECOMBINATION
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsMiki Y / Luo SC / Ho MC
Funding support Taiwan, 1 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-KPQ-105-TPP and AS-KPQ-109-TPP2 Taiwan
CitationJournal: Nat Commun / Year: 2023
Title: A RAD51-ADP double filament structure unveils the mechanism of filament dynamics in homologous recombination.
Authors: Shih-Chi Luo / Min-Chi Yeh / Yu-Hsiang Lien / Hsin-Yi Yeh / Huei-Lun Siao / I-Ping Tu / Peter Chi / Meng-Chiao Ho /
Abstract: ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the ...ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51-DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51-DNA filaments at 3.1 Å resolution, revealing a unique RAD51 double-filament that wraps around ssDNA. Structural analysis, supported by ATP-chase and time-resolved cryo-EM experiments, reveals a collapsing mechanism involving two four-protomer movements along ssDNA for mechanical transition between RAD51 single- and double-filament without RAD51 dissociation. This mechanism enables elastic change of RAD51 filament length during structural transitions between ATP- and ADP-states.
History
DepositionApr 27, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36040.png

Downloads & links

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Map

FileDownload / File: emd_36040.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a composite map with a high-resolution map (3.14 A) of the central part of the helix combined with the other part from a lower-resolution map (3.58A).
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.334
Minimum - Maximum-1.8871582 - 3.8003268
Average (Standard dev.)0.015139842 (±0.082339995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: This is the consensus low-resolution density map (3.58...

Fileemd_36040_additional_1.map
AnnotationThis is the consensus low-resolution density map (3.58 A) used for composite map generation with a high-resolution map (3.14 A) locally refined in the central part of the helix.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: This is half map A of the consensus map.

Fileemd_36040_half_map_1.map
AnnotationThis is half map A of the consensus map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: This is half map B of the consensus map.

Fileemd_36040_half_map_2.map
AnnotationThis is half map B of the consensus map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : hRAD51-ADP filament intermediate

EntireName: hRAD51-ADP filament intermediate
Components
  • Complex: hRAD51-ADP filament intermediate
    • Protein or peptide: DNA repair protein RAD51 homolog 1

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Supramolecule #1: hRAD51-ADP filament intermediate

SupramoleculeName: hRAD51-ADP filament intermediate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: hRAD51 protein filament assembled in the presence of ADP and single strand DNA
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA repair protein RAD51 homolog 1

MacromoleculeName: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ...String:
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ERLLAVAERY GLSGSDVLDN VAYARAFNTD HQTQLLYQAS AMMVESRYAL LIVDSATALY RTDYSGRGEL SA RQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQVDGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 9.8 Å
Applied symmetry - Helical parameters - Δ&Phi: 155.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2622222
Startup modelType of model: INSILICO MODEL
Final angle assignmentType: NOT APPLICABLE

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