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Open data
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Basic information
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Title | Human RADX tetramer bound to ssDNA | |||||||||
![]() | RADX tetramer bound to ssDNA dT25 | |||||||||
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![]() | oligomer OB-fold / RAD51 regulator / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex | |||||||||
Function / homology | ![]() 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 | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.0 Å | |||||||||
![]() | Balakrishnan S / Chazin WJ | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 447 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 18.2 KB 18.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 16.9 KB | Display | ![]() |
Images | ![]() | 74.8 KB | ||
Filedesc metadata | ![]() | 6.5 KB | ||
Others | ![]() ![]() | 474.4 MB 474.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8u61MC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | RADX tetramer bound to ssDNA dT25 | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.818 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: half map A
File | emd_41940_half_map_1.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map B
File | emd_41940_half_map_2.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : RADX tetramer bound to ssDNA dT25
Entire | Name: RADX tetramer bound to ssDNA dT25 |
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Components |
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-Supramolecule #1: RADX tetramer bound to ssDNA dT25
Supramolecule | Name: RADX tetramer bound to ssDNA dT25 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 600 KDa |
-Macromolecule #1: RPA-related protein RADX
Macromolecule | Name: RPA-related protein RADX / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 97.680094 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: MSGESGQPEA GPSHAGLDWP NPERNRAGVP GGVIRRAGSQ GPRSWIQKVL EQIMDSPRQC VTPSEVVPVT VLAVQRYLLE DEPRDTVPK PPLYCYDVTI SDGVYQEKCY LDPSLNSLVY QNILKVGIQM RISRVSCLYN EKRIGQGILC IDNVHCGETS D SISLETPF ...String: MSGESGQPEA GPSHAGLDWP NPERNRAGVP GGVIRRAGSQ GPRSWIQKVL EQIMDSPRQC VTPSEVVPVT VLAVQRYLLE DEPRDTVPK PPLYCYDVTI SDGVYQEKCY LDPSLNSLVY QNILKVGIQM RISRVSCLYN EKRIGQGILC IDNVHCGETS D SISLETPF RNRAHQEKPE RPLRGGKSHY LALWNNEDPY GDIWLTDKQP EEHNFSDTKI ISLSHLEMTW TNRRNFPALL VR ILHKSKL RYYGKPDKKM IEPYQTFLEV ADSSGTVSVI MWNALCPEWY KSLRVGLVLL LQDYSVKKSY PFRIQPVPVD PQI KLISTM EICLNLRDPP TNIIIIPEKQ VKPEWRLPKL NHRFTTRSEL DDMPENCICD VIGLLVFVGR VQRSKKKENR EDFW SYRWI HIADGTSEQP FIVELFSTSQ PEIFENIYPM AYFVCTQLKV VRNDNQVPKL LYLTTTNESG VFITGHRGQP YTYDA KVKN FIQWIRTKSD SGEQKNMVIG GYYPYPPVPE TFSKYSSSIK VESLLTAISE VRKEIEDLQY REQKRIAIQG IITAIK YIP HSSATESASA SETLRNANRP STSQAARVEI QERNGKRHQD DEPVNSQYFQ TTSTNLSLSN KIRILQGPHA NPVAVPQ PG ASVQTKGIKP GMPSIFNRRA NINANLQGKA RKTISDRWES QLWREKKFGL IDHLHYSRVY PESIPRKFMF EHRKFLSD Q YNSQPAKYVP PEGRPPKLDD FKSARSLGHF EVTILGLNHE IAIDVAFLPM YCPEDIRTSQ IDTLLTSMNY SCAYPQDTT GNDRLPGPRA VAGDIIKAAT ELDRVHIVGI LDICNLGNNK VEVYLHKIYS PENTS UniProtKB: RPA-related protein RADX |
-Macromolecule #2: dT25 DNA (25-MER)
Macromolecule | Name: dT25 DNA (25-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 7.559863 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT) |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.145 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV | ||||||||||||||||||
Details | Sample was cross-linked using BS3 |
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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: ![]() |
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 |
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Image processing
-Atomic model buiding 1
Refinement | Protocol: AB INITIO MODEL |
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Output model | ![]() PDB-8u61: |