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- EMDB-42162: RADX trimer consensus map -

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Basic information

Entry
Database: EMDB / ID: EMD-42162
TitleRADX trimer consensus map
Map dataRADX trimer consensus map with homogenous refinement
Sample
  • Complex: RADX trimer bound to ssDNA dT25
Keywordsoligomer OB-fold / RAD51 regulator / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsBalakrishnan S / Chazin WJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R35 GM118089, P01 CA092584 United States
CitationJournal: bioRxiv / Year: 2023
Title: Structure of RADX and mechanism for regulation of RAD51 nucleofilaments.
Authors: Swati Balakrishnan / Madison Adolph / Miaw-Sheue Tsai / Kaitlyn Gallagher / David Cortez / Walter J Chazin /
Abstract: Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of ...Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed ssDNA. To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined by cryo-electron microscopy (EM) from maps in the 2-3 Å range. The structure reveals the molecular basis for RADX oligomerization and binding of ssDNA binding. The binding of RADX to RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the growing end of RAD51 filaments.
History
DepositionOct 1, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateOct 18, 2023-
Current statusOct 18, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42162.png

Downloads & links

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Map

FileDownload / File: emd_42162.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRADX trimer consensus map with homogenous refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.102
Minimum - Maximum-0.21708682 - 0.48173362
Average (Standard dev.)-0.000050849074 (±0.010078128)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: RADX trimer homogenous refinement half map A

Fileemd_42162_half_map_1.map
AnnotationRADX trimer homogenous refinement half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RADX trimer homogenous refinement half map B

Fileemd_42162_half_map_2.map
AnnotationRADX trimer homogenous refinement half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RADX trimer bound to ssDNA dT25

EntireName: RADX trimer bound to ssDNA dT25
Components
  • Complex: RADX trimer bound to ssDNA dT25

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Supramolecule #1: RADX trimer bound to ssDNA dT25

SupramoleculeName: RADX trimer bound to ssDNA dT25 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 450 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

Concentration0.145 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
500.0 mMNaClsodium chloride
5.0 %C3H8O3glycerol
0.3 mMC7H7FO2SPMSF
2.5 mMC2H6OSBME
VitrificationCryogen name: HELIUM / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was cross-linked using BS3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 48.38 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: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 400000
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 143662
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 150000 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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