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- EMDB-29917: Cryo-EM structure of a human BCDX2/ssDNA complex -

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Entry
Database: EMDB / ID: EMD-29917
TitleCryo-EM structure of a human BCDX2/ssDNA complex
Map data
Sample
  • Complex: Cryo-EM structure of heterotetrameric human BCDX2 in complex with ssDNA
    • Protein or peptide: DNA repair protein RAD51 homolog 2
    • Protein or peptide: DNA repair protein RAD51 homolog 3
    • Protein or peptide: DNA repair protein RAD51 homolog 4
    • Protein or peptide: DNA repair protein XRCC2
    • DNA: DNA (5'-D(P*CP*CP*CP*CP*CP*C)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsDNA binding protein / ATP binding domain / homologous recombination / RAD51 paralog / RECOMBINATION / ssDNA
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 / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination ...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 / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination / gamma-tubulin binding / regulation of fibroblast apoptotic process / reciprocal meiotic recombination / centrosome cycle / 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) / sister chromatid cohesion / Resolution of D-loop Structures through Holliday Junction Intermediates / positive regulation of neurogenesis / ATP-dependent DNA damage sensor activity / male meiosis I / microtubule organizing center / Presynaptic phase of homologous DNA pairing and strand exchange / ATP-dependent activity, acting on DNA / response to X-ray / somitogenesis / interstrand cross-link repair / positive regulation of G2/M transition of mitotic cell cycle / telomere maintenance / replication fork / meiotic cell cycle / neurogenesis / 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 / cell junction / mitotic cell cycle / single-stranded DNA binding / 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 / intracellular membrane-bounded organelle / DNA repair / 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 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
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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsJia L / Wasmuth EV / Ruben EA / Sung P / Rawal Y / Greene EC / Meir A / Olsen SK
Funding support United States, 9 items
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
DepositionFeb 26, 2023-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29917.png

Downloads & links

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Map

FileDownload / File: emd_29917.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 256 pix.
= 222.72 Å		0.87 Å/pix.
x 256 pix.
= 222.72 Å		0.87 Å/pix.
x 256 pix.
= 222.72 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-1.3394945 - 2.014738
Average (Standard dev.)0.00006925882 (±0.04414338)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 222.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29917_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: #2

Fileemd_29917_half_map_1.map
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Histogram (log scale)

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Half map: #1

Fileemd_29917_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of heterotetrameric human BCDX2 in complex with...

EntireName: Cryo-EM structure of heterotetrameric human BCDX2 in complex with ssDNA
Components
  • Complex: Cryo-EM structure of heterotetrameric human BCDX2 in complex with ssDNA
    • Protein or peptide: DNA repair protein RAD51 homolog 2
    • Protein or peptide: DNA repair protein RAD51 homolog 3
    • Protein or peptide: DNA repair protein RAD51 homolog 4
    • Protein or peptide: DNA repair protein XRCC2
    • DNA: DNA (5'-D(P*CP*CP*CP*CP*CP*C)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Cryo-EM structure of heterotetrameric human BCDX2 in complex with...

SupramoleculeName: Cryo-EM structure of heterotetrameric human BCDX2 in complex with ssDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA repair protein RAD51 homolog 2

MacromoleculeName: DNA repair protein RAD51 homolog 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.125855 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGSKKLKRVG LSQELCDRLS RHQILTCQDF LCLSPLELMK VTGLSYRGVH ELLCMVSRAC APKMQTAYGI KAQRSADFSP AFLSTTLSA LDEALHGGVA CGSLTEITGP PGCGKTQFCI MMSILATLPT NMGGLEGAVV YIDTESAFSA ERLVEIAESR F PRYFNTEE ...String:
MGSKKLKRVG LSQELCDRLS RHQILTCQDF LCLSPLELMK VTGLSYRGVH ELLCMVSRAC APKMQTAYGI KAQRSADFSP AFLSTTLSA LDEALHGGVA CGSLTEITGP PGCGKTQFCI MMSILATLPT NMGGLEGAVV YIDTESAFSA ERLVEIAESR F PRYFNTEE KLLLTSSKVH LYRELTCDEV LQRIESLEEE IISKGIKLVI LDSVASVVRK EFDAQLQGNL KERNKFLARE AS SLKYLAE EFSIPVILTN QITTHLSGAL ASQADLVSPA DDLSLSEGTS GSSCVIAALG NTWSHSVNTR LILQYLDSER RQI LIAKSP LAPFTSFVYT IKEEGLVLQA YGNSHHHHHH

UniProtKB: DNA repair protein RAD51 homolog 2

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Macromolecule #2: DNA repair protein RAD51 homolog 3

MacromoleculeName: DNA repair protein RAD51 homolog 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.244609 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MRGKTFRFEM QRDLVSFPLS PAVRVKLVSA GFQTAEELLE VKPSELSKEV GISKAEALET LQIIRRECLT NKPRYAGTSE SHKKCTALE LLEQEHTQGF IITFCSALDD ILGGGVPLMK TTEICGAPGV GKTQLCMQLA VDVQIPECFG GVAGEAVFID T EGSFMVDR ...String:
MRGKTFRFEM QRDLVSFPLS PAVRVKLVSA GFQTAEELLE VKPSELSKEV GISKAEALET LQIIRRECLT NKPRYAGTSE SHKKCTALE LLEQEHTQGF IITFCSALDD ILGGGVPLMK TTEICGAPGV GKTQLCMQLA VDVQIPECFG GVAGEAVFID T EGSFMVDR VVDLATACIQ HLQLIAEKHK GEEHRKALED FTLDNILSHI YYFRCRDYTE LLAQVYLLPD FLSEHSKVRL VI VDGIAFP FRHDLDDLSL RTRLLNGLAQ QMISLANNHR LAVILTNQMT TKIDRNQALL VPALGESWGH AATIRLIFHW DRK QRLATL YKSPSQKECT VLFQIKPQGF RDTVVTSACS LQTEGSLSTR KRSRDPEEEL

UniProtKB: DNA repair protein RAD51 homolog 3

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Macromolecule #3: DNA repair protein RAD51 homolog 4

MacromoleculeName: DNA repair protein RAD51 homolog 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.084254 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAL VALRRVLLAQ FSAFPVNGAD LYEELKTSTA ILSTGIGSL DKLLDAGLYT GEVTEIVGGP GSGKTQVCLC MAANVAHGLQ QNVLYVDSNG GLTASRLLQL LQAKTQDEEE Q AEALRRIQ ...String:
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAL VALRRVLLAQ FSAFPVNGAD LYEELKTSTA ILSTGIGSL DKLLDAGLYT GEVTEIVGGP GSGKTQVCLC MAANVAHGLQ QNVLYVDSNG GLTASRLLQL LQAKTQDEEE Q AEALRRIQ VVHAFDIFQM LDVLQELRGT VAQQVTGSSG TVKVVVVDSV TAVVSPLLGG QQREGLALMM QLARELKTLA RD LGMAVVV TNHITRDRDS GRLKPALGRS WSFVPSTRIL LDTIEGAGAS GGRRMACLAK SSRQPTGFQE MVDIGTWGTS EQS ATLQGD QT

UniProtKB: DNA repair protein RAD51 homolog 4

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Macromolecule #4: DNA repair protein XRCC2

MacromoleculeName: DNA repair protein XRCC2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.992488 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MCSAFHRAES GTELLARLEG RSSLKEIEPN LFADEDSPVH GDILEFHGPE GTGKTEMLYH LTARCILPKS EGGLEVEVLF IDTDYHFDM LRLVTILEHR LSQSSEEIIK YCLGRFFLVY CSSSTHLLLT LYSLESMFCS HPSLCLLILD SLSAFYWIDR V NGGESVNL ...String:
MCSAFHRAES GTELLARLEG RSSLKEIEPN LFADEDSPVH GDILEFHGPE GTGKTEMLYH LTARCILPKS EGGLEVEVLF IDTDYHFDM LRLVTILEHR LSQSSEEIIK YCLGRFFLVY CSSSTHLLLT LYSLESMFCS HPSLCLLILD SLSAFYWIDR V NGGESVNL QESTLRKCSQ CLEKLVNDYR LVLFATTQTI MQKASSSSEE PSHASRRLCD VDIDYRPYLC KAWQQLVKHR MF FSKQDDS QSSNQFSLVS RCLKSNSLKK HFFIIGESGV EFCDYKDDDD K

UniProtKB: DNA repair protein XRCC2

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Macromolecule #5: DNA (5'-D(P*CP*CP*CP*CP*CP*C)-3')

MacromoleculeName: DNA (5'-D(P*CP*CP*CP*CP*CP*C)-3') / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.630495 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC) (DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC) (DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC) (DC)

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Macromolecule #6: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 3 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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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

Concentration0.9 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 51.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold models of BCDX2 subunits
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 165968
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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