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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 / 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 / random inactivation of X chromosome / nuclear ubiquitin ligase complex / ubiquitin-modified histone reader activity / chordate embryonic development / negative regulation of intracellular estrogen receptor signaling pathway / 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 / homologous recombination / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / tissue homeostasis / regulation of phosphorylation / protein K6-linked ubiquitination / lateral element / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / 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 / Resolution of D-loop Structures through Holliday Junction Intermediates / MicroRNA (miRNA) biogenesis / HDR through Single Strand Annealing (SSA) / intracellular membraneless organelle / 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 / 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 / mRNA Capping / mitotic G2 DNA damage checkpoint signaling / Transcriptional Regulation by E2F6 / mRNA Splicing - Minor Pathway / PIWI-interacting RNA (piRNA) biogenesis / negative regulation of cell cycle / negative regulation of reactive oxygen species metabolic process / Presynaptic phase of homologous DNA pairing and strand exchange / Processing of Capped Intron-Containing Pre-mRNA / positive regulation of vascular endothelial growth factor production / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / RNA polymerase II transcribes snRNA genes / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / ubiquitin ligase complex / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / Tat-mediated elongation of the HIV-1 transcript / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / Formation of HIV-1 elongation complex containing HIV-1 Tat / Constitutive Signaling by NOTCH1 HD Domain Mutants / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation
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 / : / Ankyrin repeat / DNA-directed RNA polymerase, subunit beta-prime / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin conserved site / RNA polymerase Rpb1, domain 3 superfamily / Ankyrin repeat-containing domain superfamily / Zinc finger RING-type profile. / 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 / 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 / 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 / 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
  • Download
  • 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

400_6340.gif

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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.5 Å/pix.
x 60 pix.
= 330. Å		5.5 Å/pix.
x 60 pix.
= 330. Å		5.5 Å/pix.
x 60 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.5 Å
Density

Histogram

Histogram (log scale)
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
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
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 54500 / 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: 50000
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.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 22000
Final two d classificationNumber classes: 5

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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:

1jnx


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:

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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