+Open data
-Basic information
Entry | Database: PDB / ID: 8u5y | ||||||
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Title | human RADX trimer bound to ssDNA | ||||||
Components |
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Keywords | DNA 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 | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å | ||||||
Authors | Balakrishnan, S. / Chazin, W.J. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8u5y.cif.gz | 809.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8u5y.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8u5y.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8u5y_validation.pdf.gz | 883.9 KB | Display | wwPDB validaton report |
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Full document | 8u5y_full_validation.pdf.gz | 897.5 KB | Display | |
Data in XML | 8u5y_validation.xml.gz | 64.8 KB | Display | |
Data in CIF | 8u5y_validation.cif.gz | 99.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u5/8u5y ftp://data.pdbj.org/pub/pdb/validation_reports/u5/8u5y | HTTPS FTP |
-Related structure data
Related structure data | 41939MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 | | Mass: 7559.863 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: RADX trimer bound to ssDNA dT25 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.45 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Homo sapiens (human) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.145 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was cross-linked using BS3 | ||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: HELIUM / Humidity: 100 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 48.38 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||
Particle selection | Num. of particles selected: 400000 | |||||||||||||||||||||
3D reconstruction | Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143622 / Symmetry type: POINT | |||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL |