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- PDB-8faz: Cryo-EM structure of the human BCDX2 complex -

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Basic information

Entry
Database: PDB / ID: 8faz
TitleCryo-EM structure of the human BCDX2 complex
Components
  • (DNA repair protein RAD51 homolog ...) x 3
  • DNA repair protein XRCC2
KeywordsRECOMBINATION / DNA binding protein / ATP binding domain / homologous recombination / RAD51 paralog
Function / homology
Function and homology information


meiotic DNA recombinase assembly / Rad51C-XRCC3 complex / Rad51B-Rad51C-Rad51D-XRCC2 complex / female meiosis sister chromatid cohesion / blastocyst growth / crossover junction DNA endonuclease activity / somite development / DNA strand invasion / telomere maintenance via recombination / Impaired BRCA2 binding to PALB2 ...meiotic DNA recombinase assembly / Rad51C-XRCC3 complex / Rad51B-Rad51C-Rad51D-XRCC2 complex / female meiosis sister chromatid cohesion / blastocyst growth / crossover junction DNA endonuclease activity / somite development / DNA strand invasion / telomere maintenance via recombination / Impaired BRCA2 binding to PALB2 / gamma-tubulin binding / reciprocal meiotic recombination / regulation of fibroblast apoptotic process / sister chromatid cohesion / centrosome cycle / 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 / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / positive regulation of neurogenesis / ATP-dependent DNA damage sensor activity / male meiosis I / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / interstrand cross-link repair / somitogenesis / positive regulation of G2/M transition of mitotic cell cycle / telomere maintenance / neurogenesis / replication fork / meiotic cell cycle / response to gamma radiation / TP53 Regulates Transcription of DNA Repair Genes / double-strand break repair via homologous recombination / multicellular organism growth / HDR through Homologous Recombination (HRR) / Meiotic recombination / single-stranded DNA binding / mitotic cell cycle / cell junction / Factors involved in megakaryocyte development and platelet production / double-stranded DNA binding / spermatogenesis / DNA recombination / in utero embryonic development / negative regulation of neuron apoptotic process / chromosome, telomeric region / regulation of cell cycle / DNA repair / intracellular membrane-bounded organelle / centrosome / positive regulation of cell population proliferation / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / DNA binding / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
DNA repair protein XRCC2 / DNA repair protein RAD51 homologue 2 / : / : / RAD51D, N-terminal domain / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. ...DNA repair protein XRCC2 / DNA repair protein RAD51 homologue 2 / : / : / RAD51D, N-terminal domain / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA repair protein RAD51 homolog 2 / DNA repair protein RAD51 homolog 3 / DNA repair protein XRCC2 / DNA repair protein RAD51 homolog 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsJia, L. / Wasmuth, E.V. / Ruben, E.A. / Sung, P. / Rawal, Y. / Greene, E.C. / Meir, A. / Olsen, S.K.
Funding support United States, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM115568 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM128731 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR200030 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA168635 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R35 CA241801 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR180029 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR210023 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00 GM140264 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR220068 United States
CitationJournal: Nature / Year: 2023
Title: Structural insights into BCDX2 complex function in homologous recombination.
Authors: Yashpal Rawal / Lijia Jia / Aviv Meir / Shuo Zhou / Hardeep Kaur / Eliza A Ruben / Youngho Kwon / Kara A Bernstein / Maria Jasin / Alexander B Taylor / Sandeep Burma / Robert Hromas / ...Authors: Yashpal Rawal / Lijia Jia / Aviv Meir / Shuo Zhou / Hardeep Kaur / Eliza A Ruben / Youngho Kwon / Kara A Bernstein / Maria Jasin / Alexander B Taylor / Sandeep Burma / Robert Hromas / Alexander V Mazin / Weixing Zhao / Daohong Zhou / Elizabeth V Wasmuth / Eric C Greene / Patrick Sung / Shaun K Olsen /
Abstract: Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks. HR depends on the products of several paralogues of RAD51, including ...Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2). BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.
History
DepositionNov 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 2, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: DNA repair protein RAD51 homolog 2
C: DNA repair protein RAD51 homolog 3
D: DNA repair protein RAD51 homolog 4
X: DNA repair protein XRCC2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,9359
Polymers149,4474
Non-polymers1,4885
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, 1:1:1:1 stoichiometric complex observed on gel filtration and also mass photometry.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA repair protein RAD51 homolog ... , 3 types, 3 molecules BCD

#1: Protein DNA repair protein RAD51 homolog 2 / / R51H2 / RAD51 homolog B / Rad51B / RAD51-like protein 1


Mass: 39125.855 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51B, RAD51L1, REC2 / Production host: Trichoplusia ni (cabbage looper) / Variant (production host): BTI-Tn-5B1-4 / References: UniProt: O15315
#2: Protein DNA repair protein RAD51 homolog 3 / / R51H3 / RAD51 homolog C / RAD51-like protein 2


Mass: 42244.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51C, RAD51L2 / Production host: Trichoplusia ni (cabbage looper) / Variant (production host): BTI-Tn-5B1-4 / References: UniProt: O43502
#3: Protein DNA repair protein RAD51 homolog 4 / / R51H3 / RAD51 homolog D / RAD51-like protein 3 / TRAD


Mass: 35084.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51D, RAD51L3 / Production host: Trichoplusia ni (cabbage looper) / Variant (production host): BTI-Tn-5B1-4 / References: UniProt: O75771

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Protein , 1 types, 1 molecules X

#4: Protein DNA repair protein XRCC2 / / X-ray repair cross-complementing protein 2


Mass: 32992.488 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC2 / Production host: Trichoplusia ni (cabbage looper) / Variant (production host): BTI-Tn-5B1-4 / References: UniProt: O43543

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Non-polymers , 3 types, 5 molecules

#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#6: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Cryo-EM structure of the human heterotetrameric BCDX2 complex
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: High Five (BTI-TN-5B1-4)
Buffer solutionpH: 7.5
SpecimenConc.: 0.9 mg/ml / Embedding 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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 51 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 489073 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027444
ELECTRON MICROSCOPYf_angle_d0.42310059
ELECTRON MICROSCOPYf_dihedral_angle_d10.8782771
ELECTRON MICROSCOPYf_chiral_restr0.0371181
ELECTRON MICROSCOPYf_plane_restr0.0031259

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