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- EMDB-6340: Electron cryo-microscopy of a BRCA1-RNAPII transcriptional complex -

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Basic information

Entry
Database: EMDB / ID: EMD-6340
TitleElectron cryo-microscopy of a BRCA1-RNAPII transcriptional complex
Map dataReconstruction of BRCA1-RNAPII transcriptional complexes derived from human breast cancer cells
Sample
  • Sample: BRCA1-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: Ubiqutin
  • DNA: DNA
Keywordstranscription / DNA repair / cancer / mRNA / tumor suppressor / ubiquitin ligase
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 / cryo EM / negative staining / Resolution: 22.0 Å
AuthorsGilmore BL / Winton CE / Demmert AC / Tanner JR / Bowman S / Karageorge V / Patel K / Sheng Z / Kelly DF
Citation
Journal: Nat Struct Mol Biol / Year: 2012
Title: Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening.
Authors: Sebastian Grünberg / Linda Warfield / Steven Hahn /
Abstract: Yeast RNA polymerase II (Pol II) general transcription factor TFIIE and the TFIIH subunit Ssl2 (yeast ortholog of mammalian XPB) function in the transition of the preinitiation complex (PIC) to the ...Yeast RNA polymerase II (Pol II) general transcription factor TFIIE and the TFIIH subunit Ssl2 (yeast ortholog of mammalian XPB) function in the transition of the preinitiation complex (PIC) to the open complex. We show that the three TFIIE winged-helix (WH) domains form a heterodimer, with the Tfa1 (TFIIEα) WH binding the Pol II clamp and the Tfa2 (TFIIEβ) tandem WH domain encircling promoter DNA that becomes single-stranded in the open complex. Ssl2 lies adjacent to TFIIE, enclosing downstream promoter DNA. Unlike previous proposals, comparison of the PIC and open-complex models strongly suggests that Ssl2 promotes DNA opening by functioning as a double-stranded-DNA translocase, feeding 15 base pairs into the Pol II cleft. Right-handed threading of DNA through the Ssl2 binding groove, combined with the fixed position of upstream promoter DNA, leads to DNA unwinding and the open state.
#5: Journal: Journal of Scientific Reports
Title: A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of the Human Disease
Authors: Gilmore BL / Winton CE / Demmert AC / Tanner JR / Bowman S / Karageorge V / Patel K / Sheng Z / Kelly DF
History
DepositionMay 20, 2015-
Header (metadata) releaseJun 3, 2015-
Map releaseMay 4, 2016-
UpdateMay 4, 2016-
Current statusMay 4, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6340.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of BRCA1-RNAPII transcriptional complexes derived from human breast cancer cells
Voxel sizeX=Y=Z: 5.5 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.04934045 - 0.27780649
Average (Standard dev.)0.00200953 (±0.01158097)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.55.55.5
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-31-64
NX/NY/NZ6963129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean-0.0490.2780.002

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Supplemental data

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Sample components

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Entire : BRCA1-RNAP II transcriptional assemblies isolated from hereditary...

EntireName: BRCA1-RNAP II transcriptional assemblies isolated from hereditary breast cancer cells
Components
  • Sample: BRCA1-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: Ubiqutin
  • DNA: DNA

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Supramolecule #1000: BRCA1-RNAP II transcriptional assemblies isolated from hereditary...

SupramoleculeName: BRCA1-RNAP II transcriptional assemblies isolated from hereditary breast cancer cells
type: sample / ID: 1000
Oligomeric state: one BRCA1 molecule binds to one polymerase complex
Number unique components: 5
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 / Location in cell: 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: 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 / Location in cell: 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 / Location in cell: nucleus
Molecular weightTheoretical: 87 KDa
SequenceUniProtKB: BRCA1-associated RING domain protein 1

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Macromolecule #4: Ubiqutin

MacromoleculeName: Ubiqutin / 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 / Location in cell: nucleus
Molecular weightTheoretical: 8 KDa
SequenceUniProtKB: Polyubiquitin-C

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Macromolecule #5: DNA

MacromoleculeName: DNA / type: dna / ID: 5 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Homo sapiens (human) / synonym: human

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
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: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: GATAN CRYOPLUNGE 3
Method: Microchips were blotted for ~8 seconds using one-sided blotting.

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 54500 / 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: 50000
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 8, 2013
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (2k x 2k) / Digitization - Sampling interval: 30 µm / Number real images: 50 / Average electron dose: 5 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 5
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 22000
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 - 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 4

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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