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- EMDB-41940: Human RADX tetramer bound to ssDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-41940
TitleHuman RADX tetramer bound to ssDNA
Map dataRADX tetramer bound to ssDNA dT25
Sample
  • Complex: RADX tetramer bound to ssDNA dT25
    • Protein or peptide: RPA-related protein RADX
    • DNA: dT25 DNA (25-MER)
Keywordsoligomer 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
RPA-related protein RADX
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsBalakrishnan S / Chazin WJ
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41940.png

Downloads & links

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Map

FileDownload / File: emd_41940.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRADX tetramer bound to ssDNA dT25
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 512 pix.
= 418.816 Å		0.82 Å/pix.
x 512 pix.
= 418.816 Å		0.82 Å/pix.
x 512 pix.
= 418.816 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.818 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.067
Minimum - Maximum-0.16872042 - 0.36966905
Average (Standard dev.)-0.000012653513 (±0.009460527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 418.816 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_41940_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_41940_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RADX tetramer bound to ssDNA dT25

EntireName: RADX tetramer bound to ssDNA dT25
Components
  • Complex: RADX tetramer bound to ssDNA dT25
    • Protein or peptide: RPA-related protein RADX
    • DNA: dT25 DNA (25-MER)

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Supramolecule #1: RADX tetramer bound to ssDNA dT25

SupramoleculeName: RADX tetramer bound to ssDNA dT25 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: RPA-related protein RADX

MacromoleculeName: RPA-related protein RADX / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 97.680094 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: 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

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Macromolecule #2: dT25 DNA (25-MER)

MacromoleculeName: dT25 DNA (25-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.559863 KDa
SequenceString:
(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)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.145 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
500.0 mMNaClsodium chloride
5.0 %C3H8O3glycerol
0.3 mMC7H7FO2SPMSF
2.5 mMC2H6OSBME
GridModel: 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was cross-linked using BS3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 48.38 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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

Particle selectionNumber selected: 400000
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 58000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 100000 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8u61:
Human RADX tetramer bound to ssDNA

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