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- EMDB-3115: the P-lobe of RNA polymerase II pre-initiation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3115
Titlethe P-lobe of RNA polymerase II pre-initiation complex
Map datarefined P-lobe of RNA polymerase II pre-initiation complex
Sample
  • Sample: refined P-lobe of yeast RNA polymerase II pre-initiation complex
  • Protein or peptide: RNA polymerase II pre-initiation complex
Keywordstranscription / pre-initiation complex / RNA polymerase / TFIIE / TFIIH / TFIIB / TBP / TFIIF
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsMurakami K / Tsai K-L / Kalisman N / Bushnell DA / Asturias FJ / Kornberg RD
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Structure of an RNA polymerase II preinitiation complex.
Authors: Kenji Murakami / Kuang-Lei Tsai / Nir Kalisman / David A Bushnell / Francisco J Asturias / Roger D Kornberg /
Abstract: The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the ...The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.
History
DepositionAug 9, 2015-
Header (metadata) releaseSep 16, 2015-
Map releaseOct 28, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 140
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 140
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3115.png

Downloads & links

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Map

FileDownload / File: emd_3115.map.gz / Format: CCP4 / Size: 104.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrefined P-lobe of RNA polymerase II pre-initiation complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 304 pix.
= 399.76 Å		1.32 Å/pix.
x 304 pix.
= 399.76 Å		1.32 Å/pix.
x 304 pix.
= 399.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.315 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 110.0 / Movie #1: 140
Minimum - Maximum-54.113346100000001 - 431.188049320000005
Average (Standard dev.)4.07078362 (±20.566879270000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 399.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3151.3151.315
M x/y/z304304304
origin x/y/z0.0000.0000.000
length x/y/z399.760399.760399.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS304304304
D min/max/mean-54.113431.1884.071

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Supplemental data

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Sample components

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Entire : refined P-lobe of yeast RNA polymerase II pre-initiation complex

EntireName: refined P-lobe of yeast RNA polymerase II pre-initiation complex
Components
  • Sample: refined P-lobe of yeast RNA polymerase II pre-initiation complex
  • Protein or peptide: RNA polymerase II pre-initiation complex

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Supramolecule #1000: refined P-lobe of yeast RNA polymerase II pre-initiation complex

SupramoleculeName: refined P-lobe of yeast RNA polymerase II pre-initiation complex
type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 1.5 MDa

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Macromolecule #1: RNA polymerase II pre-initiation complex

MacromoleculeName: RNA polymerase II pre-initiation complex / type: protein_or_peptide / ID: 1 / Name.synonym: PIC / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 1.5 MDa

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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

Concentration0.3 mg/mL
BufferpH: 7.6
Details: 20 mM HEPES (pH7.6), 5 mM DTT, 2 mM Mg(OAc)2, and 40 mM KOAc
GridDetails: 3uL was transferred to Quantifoil and flash frozen in liquid ethane with a Vitrobot.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateNov 14, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 2564 / Average electron dose: 40 e/Å2
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.70 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Sparx
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: OTHER / Software - Name: Sparx / Number images used: 37328

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