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- PDB-9tyy: RAD51-ssDNA filament in complex with calcium and ATP bound by the... -

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Basic information

Entry
Database: PDB / ID: 9tyy
TitleRAD51-ssDNA filament in complex with calcium and ATP bound by the RAD54 N-terminus
Components
  • DNA
  • DNA repair and recombination protein RAD54-like
  • DNA repair protein RAD51 homolog 1
KeywordsDNA BINDING PROTEIN / RAD51 recombinase / RAD54 / filament modulation / homologous recombination
Function / homology
Function and homology information


double-strand break repair via synthesis-dependent strand annealing / presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to cisplatin ...double-strand break repair via synthesis-dependent strand annealing / presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to cisplatin / DNA translocase activity / DNA strand invasion / cellular response to hydroxyurea / mitotic recombination / cellular response to camptothecin / replication-born double-strand break repair via sister chromatid exchange / lateral element / regulation of DNA damage checkpoint / DNA strand exchange activity / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination / ATP-dependent DNA/DNA annealing activity / reciprocal meiotic recombination / single-stranded DNA helicase activity / Homologous DNA Pairing and Strand Exchange / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / HDR through Single Strand Annealing (SSA) / ATP-dependent DNA damage sensor activity / Resolution of D-loop Structures through Holliday Junction Intermediates / regulation of double-strand break repair via homologous recombination / response to ionizing radiation / nuclear chromosome / Impaired BRCA2 binding to RAD51 / Transcriptional Regulation by E2F6 / replication fork processing / Presynaptic phase of homologous DNA pairing and strand exchange / chromosome organization / response to X-ray / ATP-dependent activity, acting on DNA / interstrand cross-link repair / condensed chromosome / DNA polymerase binding / DNA helicase activity / condensed nuclear chromosome / determination of adult lifespan / cellular response to ionizing radiation / male germ cell nucleus / meiotic cell cycle / cellular response to gamma radiation / protein-DNA complex / PML body / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / Meiotic recombination / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / response to toxic substance / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / response to xenobiotic stimulus / mitochondrial matrix / DNA repair / DNA damage response / chromatin binding / centrosome / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
: / DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical ...: / DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / : / SNF2-like, N-terminal domain superfamily / Helix-hairpin-helix domain / SNF2, N-terminal / SNF2-related domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA repair protein RAD51 homolog 1 / DNA repair and recombination protein RAD54-like
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiang, P. / Zhang, X.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: bioRxiv / Year: 2026
Title: Structures and molecular mechanisms of RAD54B in modulating homologous recombination.
Abstract: Genome stability is essential for cellular viability yet constantly threatened by endogenous and exogenous DNA-damaging agents. Among these, DNA double-strand breaks (DSBs) are particularly harmful ...Genome stability is essential for cellular viability yet constantly threatened by endogenous and exogenous DNA-damaging agents. Among these, DNA double-strand breaks (DSBs) are particularly harmful and in S/G2 phases are faithfully repaired through homologous recombination (HR), a high-fidelity pathway utilising homologous sequences in sister chromatin. The RAD51 recombinase forms nucleoprotein filaments on single-stranded DNA (ssDNA) to mediate homology search, strand invasion and subsequent D-loop formation that leads to DNA synthesis and repair. The efficiency of HR depends on precise regulation of RAD51 filament dynamics by accessory factors, including RAD54 and RAD54B, which belong to the SWI2/SNF2-family DNA translocases. While RAD54 is well-characterized, RAD54B's molecular functions remain poorly understood. Here, we define RAD54B's role in HR using cryo-electron microscopy, mutagenesis, biochemical and cellular assays. We show that RAD54B stabilizes RAD51-DNA filaments, inhibits RAD51 ATPase activity, and promotes strand invasion, D-loop formation and strand exchange. The N-terminal domain (NTD) alone supports filament stabilization and strand exchange, while the C-terminal ATPase domain is required for D-loop formation. Structural and biochemical analyses reveal three RAD51-interacting sites within the NTD and a unique domain (β-domain) that bridges RAD51 protomers and contacts donor dsDNA. This β-domain also regulates RAD54B's ATPase activity and higher-order oligomer organization on dsDNA. Cellular assays reveal that the NTD RAD51-interacting sites as well as the β-domain are required for repairing camptothecin-induced DSBs by HR in human cells. Our findings uncover a modular architecture and mechanistic framework for RAD54B function in HR, highlighting its critical role in genome maintenance.
HIGHLIGHTS: cryoEM structure of RAD54B in complex with RAD51-DNA complexRAD54B uses three sites to interact with RAD51, including a previously unrecognised β-domain that bridges distal RAD51 ...HIGHLIGHTS: cryoEM structure of RAD54B in complex with RAD51-DNA complexRAD54B uses three sites to interact with RAD51, including a previously unrecognised β-domain that bridges distal RAD51 protomers.The β-domain plays multiple crucial roles including regulating filament stability, RAD54B ATPase activity and RAD54B higher order assembly on DNA.RAD54B employs a modular mechanism, with the N-terminal region engaing and stabilising RAD51 filaments, capturing of the homologous strands, whereas the ATPase motor domainrequired for homology search and strand invasion.RAD54B N-terminus and β-domain are essential for HR-mediated repair of camptothecin-induced breaks in human cells.
History
DepositionJan 21, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 15, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
O: DNA
A: DNA repair and recombination protein RAD54-like
B: DNA repair and recombination protein RAD54-like
E: DNA repair and recombination protein RAD54-like
C: DNA repair and recombination protein RAD54-like
F: DNA repair and recombination protein RAD54-like
D: DNA repair and recombination protein RAD54-like
I: DNA repair protein RAD51 homolog 1
J: DNA repair protein RAD51 homolog 1
K: DNA repair protein RAD51 homolog 1
L: DNA repair protein RAD51 homolog 1
M: DNA repair protein RAD51 homolog 1
N: DNA repair protein RAD51 homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)238,14631
Polymers234,62213
Non-polymers3,52418
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: DNA chain DNA


Mass: 5448.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein/peptide
DNA repair and recombination protein RAD54-like / RAD54 homolog / hHR54 / hRAD54


Mass: 1186.429 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD54L, RAD54A / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q92698, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#3: Protein
DNA repair protein RAD51 homolog 1 / HsRAD51 / hRAD51 / RAD51 homolog A


Mass: 37009.125 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51, RAD51A, RECA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q06609
#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RAD51 filament in complex with calcium and ATP bound by the RAD54 N-terminus
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1388876 / Symmetry type: POINT
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315611
ELECTRON MICROSCOPYf_angle_d0.50121154
ELECTRON MICROSCOPYf_dihedral_angle_d11.3942545
ELECTRON MICROSCOPYf_chiral_restr0.0382406
ELECTRON MICROSCOPYf_plane_restr0.0042658

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