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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 / histone H2AK127 ubiquitin ligase activity / histone H2AK129 ubiquitin ligase activity / Defective DNA double strand break response due to BRCA1 loss of function / Defective DNA double strand break response due to BARD1 loss of function / BRCA1-BARD1 complex / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex / microfibril binding ...negative regulation of mRNA 3'-end processing / histone H2AK127 ubiquitin ligase activity / histone H2AK129 ubiquitin ligase activity / Defective DNA double strand break response due to BRCA1 loss of function / Defective DNA double strand break response due to BARD1 loss of function / BRCA1-BARD1 complex / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex / microfibril binding / negative regulation of centriole replication / sex-chromosome dosage compensation / negative regulation of intracellular estrogen receptor signaling pathway / random inactivation of X chromosome / nuclear ubiquitin ligase complex / ubiquitin-modified histone reader activity / chordate embryonic development / cellular response to indole-3-methanol / gamma-tubulin ring complex / negative regulation of fatty acid biosynthetic process / DNA strand resection involved in replication fork processing / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / homologous recombination / lateral element / tissue homeostasis / protein K6-linked ubiquitination / Impaired BRCA2 binding to PALB2 / regulation of phosphorylation / regulation of DNA damage checkpoint / XY body / mitotic G2/M transition checkpoint / negative regulation of protein export from nucleus / centrosome cycle / RNA polymerase binding / postreplication repair / DNA repair complex / Abortive elongation of HIV-1 transcript in the absence of Tat / 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) / FGFR2 alternative splicing / MicroRNA (miRNA) biogenesis / Resolution of D-loop Structures through Holliday Junction Intermediates / intracellular membraneless organelle / HDR through Single Strand Annealing (SSA) / Viral Messenger RNA Synthesis / Signaling by FGFR2 IIIa TM / response to ionizing radiation / negative regulation of gene expression via chromosomal CpG island methylation / Impaired BRCA2 binding to RAD51 / 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 / 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 / mitotic G2 DNA damage checkpoint signaling / Transcriptional Regulation by E2F6 / mRNA Splicing - Minor Pathway / PIWI-interacting RNA (piRNA) biogenesis / negative regulation of reactive oxygen species metabolic process / Presynaptic phase of homologous DNA pairing and strand exchange / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / Processing of Capped Intron-Containing Pre-mRNA / negative regulation of cell cycle / RNA polymerase II transcribes snRNA genes / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / positive regulation of vascular endothelial growth factor production / ubiquitin ligase complex / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / regulation of DNA repair / SUMOylation of DNA damage response and repair proteins / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / RNA polymerase II, core complex / protein autoubiquitination / RNA Polymerase II Transcription Elongation / : / Formation of RNA Pol II elongation complex / Maturation of protein E / Maturation of protein E / RNA Polymerase II Pre-transcription Events / 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 Rpb1 C-terminal repeat ...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 Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / DNA-directed RNA polymerase, subunit beta-prime / Ankyrin repeat / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / Ankyrin repeat-containing domain superfamily / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily
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
  • Download
  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6400.gif

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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4 Å/pix.
x 150 pix.
= 600. Å		4 Å/pix.
x 150 pix.
= 600. Å		4 Å/pix.
x 150 pix.
= 600. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4 Å
Density

Histogram

Histogram (log scale)
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
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
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 75000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

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

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Atomic model buiding 1

Initial modelPDB ID:

4a93


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:

1ubq


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:

1jm7


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:

1jnx


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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