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- PDB-8u61: Human RADX tetramer bound to ssDNA -

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Open data


ID or keywords:

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Basic information

Entry
Database: PDB / ID: 8u61
TitleHuman RADX tetramer bound to ssDNA
Components
  • RPA-related protein RADX
  • dT25 DNA (25-MER)
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 / RPA-related protein RADX / 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: 4 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: Proc Natl Acad Sci U S A / Year: 2024
Title: Structure of RADX and mechanism for regulation of RAD51 nucleofilaments.
Authors: Swati Balakrishnan / Madison Adolph / Miaw-Sheue Tsai / Tae Akizuki / 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 single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) 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 ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with 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 end of RAD51 filaments.
History
DepositionSep 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: RPA-related protein RADX
B: RPA-related protein RADX
A: RPA-related protein RADX
C: RPA-related protein RADX
E: dT25 DNA (25-MER)


Theoretical massNumber of molelcules
Total (without water)398,2805
Polymers398,2805
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RPA-related protein RADX


Mass: 97680.094 Da / Num. of mol.: 4
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 dT25 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 tetramer bound to ssDNA dT25 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.60 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: ETHANE / 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
2EPUimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 400000
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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