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- PDB-8u5y: human RADX trimer bound to ssDNA -

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Basic information

Entry
Database: PDB / ID: 8u5y
Titlehuman RADX trimer bound to ssDNA
Components
  • DNA (25-MER)
  • RPA-related protein RADX
KeywordsDNA BINDING PROTEIN/DNA / oligomer OB-fold / RAD51 regulator / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


negative regulation of double-strand break repair via homologous recombination / regulation of DNA repair / replication fork / single-stranded DNA binding / nuclear speck / RNA binding
Similarity search - Function
RPA-related protein RADX / Family of unknown function (DUF5521) / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / RPA-related protein RADX
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsBalakrishnan, S. / Chazin, W.J.
Funding support United States, 1items
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
DepositionSep 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RPA-related protein RADX
A: RPA-related protein RADX
C: RPA-related protein RADX
D: DNA (25-MER)


Theoretical massNumber of molelcules
Total (without water)300,6004
Polymers300,6004
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RPA-related protein RADX


Mass: 97680.094 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RADX / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q6NSI4
#2: DNA chain DNA (25-MER)


Mass: 7559.863 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RADX trimer bound to ssDNA dT25 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2500 mMsodium chlorideNaCl1
35 %glycerolC3H8O31
40.3 mMPMSFC7H7FO2S1
52.5 mMBMEC2H6OS1
SpecimenConc.: 0.145 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was cross-linked using BS3
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: HELIUM / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48.38 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameCategory
1Topazparticle selection
4cryoSPARCCTF correction
7Cootmodel fitting
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 400000
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143622 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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