+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-42162 | |||||||||
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Title | RADX trimer consensus map | |||||||||
Map data | RADX trimer consensus map with homogenous refinement | |||||||||
Sample |
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Keywords | oligomer OB-fold / RAD51 regulator / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.16 Å | |||||||||
Authors | Balakrishnan S / Chazin WJ | |||||||||
Funding support | United States, 1 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_42162.map.gz | 255.7 MB | EMDB map data format | |
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Header (meta data) | emd-42162-v30.xml emd-42162.xml | 14.8 KB 14.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_42162_fsc.xml | 16.9 KB | Display | FSC data file |
Images | emd_42162.png | 53.8 KB | ||
Filedesc metadata | emd-42162.cif.gz | 4.3 KB | ||
Others | emd_42162_half_map_1.map.gz emd_42162_half_map_2.map.gz | 475.5 MB 475.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-42162 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-42162 | HTTPS FTP |
-Validation report
Summary document | emd_42162_validation.pdf.gz | 1.2 MB | Display | EMDB validaton report |
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Full document | emd_42162_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | emd_42162_validation.xml.gz | 26.4 KB | Display | |
Data in CIF | emd_42162_validation.cif.gz | 34.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-42162 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-42162 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_42162.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | RADX trimer consensus map with homogenous refinement | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.82 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: RADX trimer homogenous refinement half map A
File | emd_42162_half_map_1.map | ||||||||||||
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Annotation | RADX trimer homogenous refinement half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: RADX trimer homogenous refinement half map B
File | emd_42162_half_map_2.map | ||||||||||||
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Annotation | RADX trimer homogenous refinement half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : RADX trimer bound to ssDNA dT25
Entire | Name: RADX trimer bound to ssDNA dT25 |
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Components |
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-Supramolecule #1: RADX trimer bound to ssDNA dT25
Supramolecule | Name: RADX trimer bound to ssDNA dT25 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 450 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.145 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.5 Component:
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Vitrification | Cryogen name: HELIUM / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV | ||||||||||||||||||
Details | Sample was cross-linked using BS3 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 48.38 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: AB INITIO MODEL |
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