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- PDB-9i62: CryoEM structure of a RAD51 D-loop -

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Basic information

Entry
Database: PDB / ID: 9i62
TitleCryoEM structure of a RAD51 D-loop
Components
  • (DNA (41-MER)) x 2
  • DNA (26-MER)
  • DNA repair protein RAD51 homolog 1
KeywordsDNA BINDING PROTEIN / DNA REPAIR / HOMOLOGOUS RECOMBINATION / STRAND EXCHANGE / ATPASE
Function / homology
Function and homology information


presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to camptothecin / 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 ...presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to camptothecin / 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 strand invasion / cellular response to hydroxyurea / mitotic recombination / DNA strand exchange activity / replication-born double-strand break repair via sister chromatid exchange / lateral element / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination / single-stranded DNA helicase activity / reciprocal meiotic recombination / 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) / Resolution of D-loop Structures through Holliday Junction Intermediates / HDR through Single Strand Annealing (SSA) / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / nuclear chromosome / Impaired BRCA2 binding to RAD51 / Transcriptional Regulation by E2F6 / replication fork processing / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / interstrand cross-link repair / condensed chromosome / DNA polymerase binding / condensed nuclear chromosome / cellular response to ionizing radiation / meiotic cell cycle / male germ cell nucleus / cellular response to gamma radiation / double-strand break repair via homologous recombination / PML body / HDR through Homologous Recombination (HRR) / response to toxic substance / Meiotic recombination / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / response to xenobiotic stimulus / DNA repair / DNA damage response / centrosome / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / protein-containing complex / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP 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 / Helix-hairpin-helix domain ...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 / Helix-hairpin-helix 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
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsPellegrini, L. / Joudeh, L. / Appleby, R.E.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust221892/Z/20/Z United Kingdom
UK Research and Innovation (UKRI)2271086 United Kingdom
Citation
Journal: Elife / Year: 2025
Title: Structural mechanism of strand exchange by the RAD51 filament.
Authors: Luay Joudeh / Robert E Appleby / Joseph D Maman / Luca Pellegrini /
Abstract: Homologous recombination (HR) preserves genomic stability by repairing double-strand DNA breaks and ensuring efficient DNA replication. Central to HR is the strand-exchange reaction taking place ...Homologous recombination (HR) preserves genomic stability by repairing double-strand DNA breaks and ensuring efficient DNA replication. Central to HR is the strand-exchange reaction taking place within the three-stranded synapsis wherein a RAD51 nucleoprotein filament binds to a donor DNA. Here, we present the cryoEM structure of a displacement loop of human RAD51 that captures the synaptic state when the filament has become tightly bound to the donor DNA. The structure elucidates the mechanism of strand exchange by RAD51, including the filament engagement with the donor DNA, the strand invasion and pairing with the complementary sequence of the donor DNA, the capture of the non-complementary strand and the polarity of the strand-exchange reaction. Our findings provide fundamental mechanistic insights into the biochemical reaction of eukaryotic HR.
#1: Journal: Elife / Year: 2025
Title: Structural mechanism of strand exchange by the RAD51 filament
Authors: Joudeh, L. / Appleby, R.E. / Maman, J.D. / Pellegrini, L.
History
DepositionJan 29, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA repair protein RAD51 homolog 1
B: DNA repair protein RAD51 homolog 1
C: DNA repair protein RAD51 homolog 1
D: DNA repair protein RAD51 homolog 1
E: DNA repair protein RAD51 homolog 1
F: DNA repair protein RAD51 homolog 1
G: DNA repair protein RAD51 homolog 1
H: DNA repair protein RAD51 homolog 1
I: DNA repair protein RAD51 homolog 1
J: DNA (26-MER)
K: DNA (41-MER)
L: DNA (41-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)378,88339
Polymers373,59712
Non-polymers5,28627
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

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Protein , 1 types, 9 molecules ABCDEFGHI

#1: Protein
DNA repair protein RAD51 homolog 1 / HsRAD51 / hRAD51 / RAD51 homolog A


Mass: 37009.125 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Details: RAD51 protomers A, B, C, D, I contain amino acids 21 to 273 and 282 to 339.
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51, RAD51A, RECA / Production host: Escherichia coli (E. coli) / References: UniProt: Q06609

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DNA chain , 3 types, 3 molecules JKL

#2: DNA chain DNA (26-MER)


Mass: 9768.257 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (41-MER)


Mass: 15271.798 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (41-MER)


Mass: 15474.897 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 27 molecules

#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Complex of RAD51 pre-synaptic filament with donor DNA.
Type: COMPLEX
Details: Donor DNA contains a 'mismatch bubble' with a complementary sequence to the invading sequence of the filament.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 900 nm
Image recordingAverage exposure time: 1.34 sec. / Electron dose: 48.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8834

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.4.1CTF correction
7Coot0.9.8.94model fitting
9cryoSPARC4.4.1initial Euler assignment
10cryoSPARC4.4.1final Euler assignment
12cryoSPARC4.4.13D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 102679 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
RefinementHighest resolution: 2.64 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00624785
ELECTRON MICROSCOPYf_angle_d0.44133980
ELECTRON MICROSCOPYf_dihedral_angle_d14.8889720
ELECTRON MICROSCOPYf_chiral_restr0.0393861
ELECTRON MICROSCOPYf_plane_restr0.0033992

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