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- EMDB-6400: Electron microscopy of BRCA1(5832insC) mutant-RNAP II transcripti... -

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Basic information

Entry
Database: EMDB / ID: EMD-6400
TitleElectron microscopy of BRCA1(5832insC) mutant-RNAP II transcriptional complex
Map dataReconstruction of BRCA1-RNAP II mutant complex
Sample
  • Sample: Mutant BRCA1(5382insC)-RNAP II transcriptional assemblies isolated from hereditary breast cancer cells
  • Protein or peptide: RNA Polyermase II core
  • Protein or peptide: BRCA1
  • Protein or peptide: BARD1
  • Protein or peptide: Ubiquitin
Keywordstranscription / DNA damage repair / breast cancer / protein
Function / homology
Function and homology information


negative regulation of mRNA 3'-end processing / Defective DNA double strand break response due to BRCA1 loss of function / Defective DNA double strand break response due to BARD1 loss of function / : / microfibril binding / BRCA1-BARD1 complex / : / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex ...negative regulation of mRNA 3'-end processing / Defective DNA double strand break response due to BRCA1 loss of function / Defective DNA double strand break response due to BARD1 loss of function / : / microfibril binding / BRCA1-BARD1 complex / : / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex / random inactivation of X chromosome / negative regulation of centriole replication / gamma-tubulin ring complex / negative regulation of intracellular estrogen receptor signaling pathway / nuclear ubiquitin ligase complex / DNA strand resection involved in replication fork processing / chordate embryonic development / negative regulation of fatty acid biosynthetic process / cellular response to indole-3-methanol / homologous recombination / lateral element / tissue homeostasis / XY body / protein K6-linked ubiquitination / regulation of DNA damage checkpoint / regulation of phosphorylation / dosage compensation by inactivation of X chromosome / negative regulation of gene expression via chromosomal CpG island methylation / Impaired BRCA2 binding to PALB2 / : / mitotic G2/M transition checkpoint / negative regulation of protein export from nucleus / postreplication repair / Abortive elongation of HIV-1 transcript in the absence of Tat / DNA repair complex / FGFR2 alternative splicing / RNA polymerase binding / 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) / MicroRNA (miRNA) biogenesis / Resolution of D-loop Structures through Holliday Junction Intermediates / Viral Messenger RNA Synthesis / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / intracellular non-membrane-bounded organelle / Signaling by FGFR2 IIIa TM / response to ionizing radiation / DNA-binding transcription activator activity / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / Formation of the HIV-1 Early Elongation Complex / mRNA Capping / mRNA Splicing - Minor Pathway / PIWI-interacting RNA (piRNA) biogenesis / HIV Transcription Initiation / RNA Polymerase II HIV Promoter Escape / Transcription of the HIV genome / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / Transcriptional Regulation by E2F6 / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / mitotic G2 DNA damage checkpoint signaling / Presynaptic phase of homologous DNA pairing and strand exchange / Processing of Capped Intron-Containing Pre-mRNA / RNA polymerase II activity / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / RNA polymerase II transcribes snRNA genes / negative regulation of cell cycle / positive regulation of vascular endothelial growth factor production / Tat-mediated elongation of the HIV-1 transcript / negative regulation of reactive oxygen species metabolic process / Formation of HIV-1 elongation complex containing HIV-1 Tat / localization / Formation of HIV elongation complex in the absence of HIV Tat / regulation of DNA repair / RNA polymerase II, core complex / protein autoubiquitination / SUMOylation of DNA damage response and repair proteins / ubiquitin ligase complex / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis
Similarity search - Function
BARD1, Zinc finger, RING-type / zf-RING of BARD1-type protein / Breast cancer type 1 susceptibility protein (BRCA1) / BRCA1, serine-rich domain / BRCA1-associated / Serine-rich domain associated with BRCT / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / BRCA1 C Terminus (BRCT) domain / RNA polymerase II, heptapeptide repeat, eukaryotic ...BARD1, Zinc finger, RING-type / zf-RING of BARD1-type protein / Breast cancer type 1 susceptibility protein (BRCA1) / BRCA1, serine-rich domain / BRCA1-associated / Serine-rich domain associated with BRCT / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / BRCA1 C Terminus (BRCT) domain / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / breast cancer carboxy-terminal domain / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Ring finger / Ankyrin repeats (3 copies) / RNA polymerase Rpb1, domain 3 superfamily / Ankyrin repeat profile. / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / Ankyrin repeat region circular profile. / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / ankyrin repeats / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / Ankyrin repeat / Zinc finger RING-type profile. / Ankyrin repeat-containing domain superfamily / Zinc finger, RING-type / Ubiquitin conserved site / Ubiquitin domain signature. / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Polyubiquitin-C / DNA-directed RNA polymerase II subunit RPB1 / Breast cancer type 1 susceptibility protein / BRCA1-associated RING domain protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsWinton CE / Gilmore BL / Demmert AC / Tanner JR / Bowman S / Karageorge V / Patel K / Sheng Z / Kelly DF
Citation
Journal: Nat Struct Biol / Year: 2001
Title: Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1.
Authors: R S Williams / R Green / J N Glover /
Abstract: The C-terminal BRCT region of BRCA1 is essential for its DNA repair, transcriptional regulation and tumor suppressor functions. Here we determine the crystal structure of the BRCT domain of human ...The C-terminal BRCT region of BRCA1 is essential for its DNA repair, transcriptional regulation and tumor suppressor functions. Here we determine the crystal structure of the BRCT domain of human BRCA1 at 2.5 A resolution. The domain contains two BRCT repeats that adopt similar structures and are packed together in a head-to-tail arrangement. Cancer-causing missense mutations occur at the interface between the two repeats and destabilize the structure. The manner by which the two BRCT repeats interact in BRCA1 may represent a general mode of interaction between homologous domains within proteins that interact to regulate the cellular response to DNA damage. The structure provides a basis to predict the structural consequences of uncharacterized BRCA1 mutations.
#3: Journal: NAT.STRUCT.BIOL. / Year: 2001
Title: Structure of a BRCA1-BARD1 heterodimeric RING-RING complex
Authors: Brzovic PS / Rajagopal P / Hoyt DW / King MC / Klevit RE
#4: Journal: J.MOL.BIOL. / Year: 1987
Title: Structure of ubiquitin refined at 1.8 A resolution
Authors: Vijay-Kumar S / Bugg CE / Cook WJ
History
DepositionJul 29, 2015-
Header (metadata) releaseAug 19, 2015-
Map releaseAug 3, 2016-
UpdateSep 14, 2016-
Current statusSep 14, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6400.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of BRCA1-RNAP II mutant complex
Voxel sizeX=Y=Z: 4 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.71576554 - 1.72933328
Average (Standard dev.)0.00979374 (±0.04155805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 600.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z600.000600.000600.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.7161.7290.010

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Supplemental data

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Sample components

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Entire : Mutant BRCA1(5382insC)-RNAP II transcriptional assemblies isolate...

EntireName: Mutant BRCA1(5382insC)-RNAP II transcriptional assemblies isolated from hereditary breast cancer cells
Components
  • Sample: Mutant BRCA1(5382insC)-RNAP II transcriptional assemblies isolated from hereditary breast cancer cells
  • Protein or peptide: RNA Polyermase II core
  • Protein or peptide: BRCA1
  • Protein or peptide: BARD1
  • Protein or peptide: Ubiquitin

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Supramolecule #1000: Mutant BRCA1(5382insC)-RNAP II transcriptional assemblies isolate...

SupramoleculeName: Mutant BRCA1(5382insC)-RNAP II transcriptional assemblies isolated from hereditary breast cancer cells
type: sample / ID: 1000
Oligomeric state: one mutant BRCA1(5382insC) molecule binds to one polymerase complex
Number unique components: 4
Molecular weightTheoretical: 800 KDa

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Macromolecule #1: RNA Polyermase II core

MacromoleculeName: RNA Polyermase II core / type: protein_or_peptide / ID: 1 / Name.synonym: Pol2 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: breast / Cell: tumor / Organelle: nucleus
Molecular weightTheoretical: 500 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB1

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Macromolecule #2: BRCA1

MacromoleculeName: BRCA1 / type: protein_or_peptide / ID: 2
Details: Mutated BRCA1 was attached to tunable microchips using antibodies against the BRCA1 C-terminal domain.
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: breast / Cell: tumor / Organelle: nucleus
Molecular weightTheoretical: 200 KDa
SequenceUniProtKB: Breast cancer type 1 susceptibility protein

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Macromolecule #3: BARD1

MacromoleculeName: BARD1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: breast / Cell: tumor / Organelle: nucleus
Molecular weightTheoretical: 87 KDa
SequenceUniProtKB: BRCA1-associated RING domain protein 1

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Macromolecule #4: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: breast / Cell: tumor / Organelle: nucleus
Molecular weightTheoretical: 8 KDa
SequenceUniProtKB: Polyubiquitin-C

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 50 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2
StainingType: NEGATIVE
Details: Protein complexes were adsorbed to antibody-decorated microchips for 2 minutes and stained with 1% uranyl formate for 1 minute.
GridDetails: SiN microchips with TEM windows coated with 25% Ni-NTA functionalized lipid monolayers
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 75000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 68000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 83 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at high magnification.
Detailslow-dose illumination
DateMar 23, 2015
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (2k x 2k) / Digitization - Sampling interval: 30 µm / Number real images: 129 / Average electron dose: 5 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 1
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 3000
DetailsThe particles were selected using an automatic selection program.

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Atomic model buiding 1

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using the Chimera software package.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using the Chimera software package.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using the Chimera software package.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 4

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera
DetailsThis domain was used to generate a homology based model using the program SWISS-MODEL.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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