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- EMDB-3981: Structure of mammalian RNA polymerase II elongation complex inhib... -

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Entry
Database: EMDB / ID: 3981
TitleStructure of mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Map dataPost-processed masked EM map of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin
SampleMammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Function/homologyTP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase I Chain Elongation / RNA Polymerase II Pre-transcription Events / Transcriptional regulation by small RNAs / B-WICH complex positively regulates rRNA expression / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / Formation of the Early Elongation Complex / mRNA Capping / mRNA Splicing - Minor Pathway ...TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase I Chain Elongation / RNA Polymerase II Pre-transcription Events / Transcriptional regulation by small RNAs / B-WICH complex positively regulates rRNA expression / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / Formation of the Early Elongation Complex / mRNA Capping / mRNA Splicing - Minor Pathway / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase II Promoter Escape / Formation of RNA Pol II elongation complex / RNA Polymerase III Transcription Initiation From Type 3 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase I Transcription Termination / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Elongation / mRNA Splicing - Major Pathway / Formation of TC-NER Pre-Incision Complex / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / nuclear-transcribed mRNA catabolic process, exonucleolytic / RNA polymerase II, Rpb4, core / RNA polymerase II, Rpb4 / maintenance of transcriptional fidelity during DNA-templated transcription elongation from RNA polymerase II promoter / RNA polymerase RBP11 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase Rpb7, N-terminal / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 6 / RNA polymerase II, heptapeptide repeat, eukaryotic / positive regulation of translational initiation / Eukaryotic RNA polymerase II heptapeptide repeat. / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / HRDC-like superfamily / Zinc finger TFIIS-type signature. / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / DNA-directed RNA polymerase RPB5 subunit, eukaryote/virus / RNA polymerase Rpb5, N-terminal domain superfamily / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb5, N-terminal / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerase, Rpb8 / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase subunit RPB10 / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / DNA-directed RNA polymerase, subunit N/Rpb10 / Archaeal RpoK/eukaryotic RPB6 RNA polymerase subunit / Zinc finger, TFIIS-type / Zinc finger TFIIS-type profile. / RNA polymerases K / 14 to 18 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / transcription by RNA polymerase I / nuclear euchromatin / RNA polymerases D / 30 to 40 Kd subunits signature. / Archaeal RpoH /eukaryotic RPB5 RNA polymerase subunit / RNA polymerases L / 13 to 16 Kd subunits signature. / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RPB5-like RNA polymerase subunit / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase, RBP11-like dimerisation domain / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase III complex / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / DNA-directed RNA polymerase I complex / RNA polymerase Rpb4 / transcription by RNA polymerase III / S1 domain / RNA-binding domain, S1 / DNA-directed RNA polymerase II, core complex / mRNA cleavage / translation initiation factor binding / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase subunit, RPB6/omega / RNA polymerase, subunit omega/K/RPB6 / RPB6/omega subunit-like superfamily / RNA polymerases M/15 Kd subunit / RNA polymerase Rpb5, N-terminal domain / RNA polymerase Rpb8 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion
Function and homology information
SourceSus scrofa / mammal / image: Sus scrofa domestica
Synthetic construct
Amanita phalloides / fungus /
Sus scrofa / mammal / image: Sus scrofa domestica
Methodsingle particle reconstruction, at 3.6 Å resolution
AuthorsLiu X / Farnung L
CitationJournal: J. Biol. Chem. / Year: 2018
Title: Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin.
Authors: Xiangyang Liu / Lucas Farnung / Christoph Wigge / Patrick Cramer
Abstract: RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step ...RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II, but forms additional contacts with metazoan-specific residues, which explain why its affinity to mammalian Pol II is ~3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.
Copyright: Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
Validation ReportPDB-ID: 6exv

SummaryFull reportAbout validation report
DateDeposition: Nov 9, 2017 / Header (metadata) release: Dec 20, 2017 / Map release: Mar 21, 2018 / Last update: Mar 28, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0577
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.0577
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6exv
  • Surface level: 0.0577
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_3981.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.07 Å/pix.
= 273.92 Å
256 pix
1.07 Å/pix.
= 273.92 Å
256 pix
1.07 Å/pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour Level:0.0577 (by author), 0.0577 (movie #1):
Minimum - Maximum-0.30933884 - 0.446918
Average (Standard dev.)0.00072788406 (0.012988935)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin000
Limit255255255
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3090.4470.001

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Supplemental data

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Mask #1

Fileemd_3981_msk_1.map ( map file in CCP4 format, 67109 KB )
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms
Data typeImage stored as Reals
Annotation detailsMask used for psot-processing
Space group number1

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Mask #1~

Fileemd_3981_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Mammalian RNA polymerase II elongation complex inhibited by Alpha...

EntireName: Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Number of components: 22
MassTheoretical: 607 kDa

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Component #1: protein, Mammalian RNA polymerase II elongation complex inhibited...

ProteinName: Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Recombinant expression: No
MassTheoretical: 607 kDa
Source (engineered)Expression System: Synthetic construct

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Component #2: protein, RNA polymerase II elongation complex

ProteinName: RNA polymerase II elongation complex / Recombinant expression: No
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #3: protein, Nucleic acids

ProteinName: Nucleic acids / Recombinant expression: No
SourceSpecies: Synthetic construct

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Component #4: protein, Alpha-amanitin

ProteinName: Alpha-amanitin / Recombinant expression: No
SourceSpecies: Amanita phalloides / fungus /

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Component #5: protein, DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1

ProteinName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1 / Recombinant expression: No
MassTheoretical: 217.450078 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #6: protein, DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2

ProteinName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2 / Recombinant expression: No
MassTheoretical: 133.201625 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #7: protein, DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3

ProteinName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3 / Recombinant expression: No
MassTheoretical: 31.439074 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #8: protein, DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3

ProteinName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3 / Recombinant expression: No
MassTheoretical: 16.331255 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #9: protein, DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1

ProteinName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
Recombinant expression: No
MassTheoretical: 24.644318 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #10: protein, DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1

ProteinName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
Recombinant expression: No
MassTheoretical: 14.477001 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #11: protein, DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7

ProteinName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7 / Recombinant expression: No
MassTheoretical: 19.314283 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #12: protein, DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3

ProteinName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3
Recombinant expression: No
MassTheoretical: 17.162273 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #13: protein, DNA-directed RNA polymerase II subunit RPB9

ProteinName: DNA-directed RNA polymerase II subunit RPB9 / Recombinant expression: No
MassTheoretical: 14.541221 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #14: protein, DNA-directed RNA polymerases I, II, and III subunit RPABC5

ProteinName: DNA-directed RNA polymerases I, II, and III subunit RPABC5
Recombinant expression: No
MassTheoretical: 7.655123 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #15: protein, DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4

ProteinName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
Recombinant expression: No
MassTheoretical: 13.310284 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #16: protein, DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4

ProteinName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
Recombinant expression: No
MassTheoretical: 7.018244 kDa
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (natural)Organ or tissue: Thymus

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Component #17: protein, AMATOXIN

ProteinName: AMATOXIN / Recombinant expression: No
MassTheoretical: 0.939004 kDa
SourceSpecies: Amanita phalloides / fungus /

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Component #18: nucleic-acid, DNA (25-MER)

Nucleic-acidName: DNA (25-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DG)(DC)(DA)(DG)(DT)(DA)(DC)(DT)(DA) (DG)(DT)(DA)(DT)(DT)(DC)(DT)(DA)(DG)(DT) (DA)(DT)(DT)(DG)(DA)(DA)(DA)(DG)(DT)(DA) (DC)(DT)(DT)(DG)(DA)(DG)(DC)(DT)(DT)(DG) (DA)(DT)(DC)
MassTheoretical: 13.303563 kDa

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Component #19: nucleic-acid, RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*...

Nucleic-acidName: RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UAUAUGCAUA AAGACCAGGC
MassTheoretical: 6.414902 kDa

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Component #20: nucleic-acid, DNA (36-MER)

Nucleic-acidName: DNA (36-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DA)(DT)(DC)(DA)(DA)(DG)(DC)(DT)(DC) (DA)(DA)(DG)(DT)(DA)(DC)(DT)(DT)(DA)(DA) (DG)(DC)(DC)(DT)(DG)(DG)(DT)(DC)(DT)(DA) (DT)(DA)(DC)(DT)(DA)(DG)(DT)(DA)(DC)(DT) (DG)(DC)(DC)
MassTheoretical: 13.178483 kDa

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Component #21: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #22: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.244 mg/ml / pH: 7.6
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 1 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 130000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 2500.0 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2264

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 134512
3D reconstructionSoftware: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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