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- PDB-9e6l: Cryo-EM structure of yeast Rad51 nucleoprotein filament bound to ... -

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Basic information

Entry
Database: PDB / ID: 9e6l
TitleCryo-EM structure of yeast Rad51 nucleoprotein filament bound to Rad54peptide
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA repair and recombination protein RAD54
  • DNA repair protein RAD51
KeywordsRECOMBINATION/DNA / Rad51 / DNA recombinase / Homologous recombination / DNA repair / RECOMBINATION / RECOMBINATION-DNA complex
Function / homology
Function and homology information


double-strand break repair via synthesis-dependent strand annealing / Presynaptic phase of homologous DNA pairing and strand exchange / heteroduplex formation / meiotic joint molecule formation / mitochondrial chromosome / mitotic recombination-dependent replication fork processing / DNA geometric change / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / DNA strand invasion ...double-strand break repair via synthesis-dependent strand annealing / Presynaptic phase of homologous DNA pairing and strand exchange / heteroduplex formation / meiotic joint molecule formation / mitochondrial chromosome / mitotic recombination-dependent replication fork processing / DNA geometric change / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / DNA strand invasion / mitotic recombination / DNA translocase activity / DNA strand exchange activity / telomere maintenance via recombination / reciprocal meiotic recombination / mitochondrial DNA repair / ATP-dependent DNA damage sensor activity / nuclear chromosome / ATP-dependent activity, acting on DNA / condensed nuclear chromosome / double-strand break repair via homologous recombination / nucleotide-excision repair / helicase activity / G2/M transition of mitotic cell cycle / double-strand break repair / single-stranded DNA binding / double-stranded DNA binding / DNA recombination / DNA helicase / chromatin remodeling / mitochondrial matrix / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Rad54, N-terminal / Rad54 N terminal / : / 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 ...Rad54, N-terminal / Rad54 N terminal / : / 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 / : / SNF2-like, N-terminal domain superfamily / 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 / DNA repair and recombination protein RAD54
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsShin, Y. / Greene, E.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for Rad54- and Hed1-mediated regulation of Rad51 during the transition from mitotic to meiotic recombination.
Authors: Yeonoh Shin / Michael T Petassi / Aidan M Jessop / Stefan Y Kim / Razvan Matei / Katherine Morse / Vivek B Raina / Upasana Roy / Eric C Greene /
Abstract: Rad51 catalyzes the DNA pairing reactions that take place during homologous recombination (HR), and HR must be tightly regulated to ensure physiologically appropriate outcomes. Rad54 is an ATP- ...Rad51 catalyzes the DNA pairing reactions that take place during homologous recombination (HR), and HR must be tightly regulated to ensure physiologically appropriate outcomes. Rad54 is an ATP-dependent DNA motor protein that stimulates Rad51 activity during mitosis. In meiosis Rad51 is downregulated by the protein Hed1, which blocks Rad54 binding to Rad51, and allows Dmc1 to function as the active recombinase. We currently have a poor understanding of the regulatory interplay between Rad54, Hed1, Rad51, and Dmc1. Here, we identify a conserved Rad51 interaction motif within Rad54, and we solve a CryoEM structure of this motif bound to Rad51. We also identify a distinct Rad51 interaction motif within Hed1 and solve its structure bound to Rad51. These structures explain how Rad54 engages Rad51 to promote recombination between sister chromatids during mitosis and how Rad51 is downregulated by Hed1 upon entry into meiosis such that its meiosis-specific homolog Dmc1 can promote recombination between homologous chromosomes.
History
DepositionOct 30, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.0Jan 22, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 22, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Jan 22, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
Revision 1.2Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: DNA repair and recombination protein RAD54
H: DNA repair and recombination protein RAD54
I: DNA repair and recombination protein RAD54
J: DNA repair and recombination protein RAD54
K: DNA repair and recombination protein RAD54
X: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
C: DNA repair protein RAD51
F: DNA repair protein RAD51
B: DNA repair protein RAD51
E: DNA repair protein RAD51
A: DNA repair protein RAD51
D: DNA repair protein RAD51
hetero molecules


Theoretical massNumber of molelcules
Total (without water)238,51430
Polymers235,18012
Non-polymers3,33518
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: Protein/peptide
DNA repair and recombination protein RAD54


Mass: 4033.730 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32863, DNA helicase
#2: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 5430.513 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#3: Protein
DNA repair protein RAD51


Mass: 34930.070 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD51, YER095W / Production host: Escherichia coli (E. coli) / References: UniProt: P25454
#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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 nucleoprotein filament bound to Rad54 peptide / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 59.51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 363379 / Symmetry type: POINT
RefinementHighest resolution: 3.3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216841
ELECTRON MICROSCOPYf_angle_d0.42722822
ELECTRON MICROSCOPYf_dihedral_angle_d10.272472
ELECTRON MICROSCOPYf_chiral_restr0.0392597
ELECTRON MICROSCOPYf_plane_restr0.0022898

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