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- EMDB-2194: human RNA Polymerase II -

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Basic information

Entry
Database: EMDB / ID: EMD-2194
Titlehuman RNA Polymerase II
Map datahuman Pol II
Sample
  • Sample: human Pol II
  • Protein or peptide: x 12 types
Keywordstranscription regulation / RNA polymerase II / non-coding RNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 25.0 Å
AuthorsKassube SA / Fang J / Grob P / Yakovchuk P / Goodrich JA / Nogales E
CitationJournal: J Mol Biol / Year: 2013
Title: Structural insights into transcriptional repression by noncoding RNAs that bind to human Pol II.
Authors: Susanne A Kassube / Jie Fang / Patricia Grob / Petro Yakovchuk / James A Goodrich / Eva Nogales /
Abstract: Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding ...Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain noncoding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. Here, we present cryo-electron microscopy reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, which is also shared with a previously characterized yeast aptamer, is close to the active center and, thus, in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNAs analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.
History
DepositionSep 11, 2012-
Header (metadata) releaseSep 26, 2012-
Map releaseJan 16, 2013-
UpdateSep 18, 2013-
Current statusSep 18, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.139
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.139
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2194.png

Downloads & links

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Map

FileDownload / File: emd_2194.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman Pol II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.01 Å/pix.
x 128 pix.
= 385.28 Å		3.01 Å/pix.
x 128 pix.
= 385.28 Å		3.01 Å/pix.
x 128 pix.
= 385.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.01 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.139 / Movie #1: 0.139
Minimum - Maximum-0.12026653 - 0.35730726
Average (Standard dev.)-0.00622475 (±0.03049673)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 385.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.013.013.01
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z385.280385.280385.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1200.357-0.006

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Supplemental data

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Sample components

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Entire : human Pol II

EntireName: human Pol II
Components
  • Sample: human Pol II
  • Protein or peptide: Rpb12
  • Protein or peptide: Rpb11
  • Protein or peptide: Rpb10
  • Protein or peptide: Rpb9
  • Protein or peptide: Rpb8
  • Protein or peptide: Rpb7
  • Protein or peptide: Rpb6
  • Protein or peptide: Rpb5
  • Protein or peptide: Rpb4
  • Protein or peptide: Rpb3
  • Protein or peptide: Rpb2
  • Protein or peptide: Rpb1

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Supramolecule #1000: human Pol II

SupramoleculeName: human Pol II / type: sample / ID: 1000 / Oligomeric state: 12 subunit complex / Number unique components: 12
Molecular weightTheoretical: 520 KDa

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Macromolecule #1: Rpb12

MacromoleculeName: Rpb12 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #2: Rpb11

MacromoleculeName: Rpb11 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #3: Rpb10

MacromoleculeName: Rpb10 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #4: Rpb9

MacromoleculeName: Rpb9 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #5: Rpb8

MacromoleculeName: Rpb8 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #6: Rpb7

MacromoleculeName: Rpb7 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #7: Rpb6

MacromoleculeName: Rpb6 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #8: Rpb5

MacromoleculeName: Rpb5 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #9: Rpb4

MacromoleculeName: Rpb4 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #10: Rpb3

MacromoleculeName: Rpb3 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 30 KDa

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Macromolecule #11: Rpb2

MacromoleculeName: Rpb2 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 130 KDa

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Macromolecule #12: Rpb1

MacromoleculeName: Rpb1 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 220 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.0312 mg/mL
BufferpH: 7.9
Details: 10 mM Tris pH 7.9, 10 mM HEPES pH 8.0, 4 mM MgCl2, 50 mM KCl, 0.05% NP-40, 1 mM DTT, 0.1% trehalose
GridDetails: 400 mesh C-flat grids (Protochips Inc.) with a thin carbon film floated on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 86 K / Instrument: FEI VITROBOT MARK II / Method: 6 sec blot, offset -2

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureAverage: 78 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateJul 10, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 1.5 µm / Number real images: 972 / Average electron dose: 20 e/Å2 / Details: data collected with leginon
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Side-entry cryostage / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, FREALIGN
Details: the data set was sorted into three different conformations using an automated multi-model approach; the initial reconstructions were used as starting models for multi-model projection ...Details: the data set was sorted into three different conformations using an automated multi-model approach; the initial reconstructions were used as starting models for multi-model projection matching in EMAN2, followed by final refinement in FREALIGN
Number images used: 8905

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