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- EMDB-2394: Architecture of an RNA polymerase II transcription pre-initiation... -

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Basic information

Entry
Database: EMDB / ID: EMD-2394
TitleArchitecture of an RNA polymerase II transcription pre-initiation complex
Map dataReconstruction of RNA polymerase II transcription pre-initiation complex
Sample
  • Sample: RNA polymerase II transcription pre-initiation complex
  • Protein or peptide: RNA polymerase II transcription pre-initiation complex
KeywordsRNA Polymerase II / Transcription / Transcription Initiation Factors / Yeast / TFIIA / TFIIB / TBP / TFIIE / TFIIF / TFIIH / TFIIS
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 16.0 Å
AuthorsMurakami K / Elmlund H / Kalisman N / Bushnell DA / Adams CM / Azubel M / Elmlund D / Levi-Kalisman Y / Liu X / Levitt M / Kornberg RD
CitationJournal: Science / Year: 2013
Title: Architecture of an RNA polymerase II transcription pre-initiation complex.
Authors: Kenji Murakami / Hans Elmlund / Nir Kalisman / David A Bushnell / Christopher M Adams / Maia Azubel / Dominika Elmlund / Yael Levi-Kalisman / Xin Liu / Brian J Gibbons / Michael Levitt / Roger D Kornberg /
Abstract: The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron ...The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.
History
DepositionJun 5, 2013-
Header (metadata) releaseJun 19, 2013-
Map releaseOct 2, 2013-
UpdateNov 20, 2013-
Current statusNov 20, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 60000
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 60000
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2394.png

Downloads & links

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Map

FileDownload / File: emd_2394.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of RNA polymerase II transcription pre-initiation complex
Voxel sizeX=Y=Z: 1.85 Å
Density
Contour LevelBy EMDB: 125000.0 / Movie #1: 60000
Minimum - Maximum-836502.8125 - 3397460.75
Average (Standard dev.)16963.73046875 (±128061.1953125)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 370.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.851.851.85
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z370.000370.000370.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-836502.8123397460.75016963.730

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Supplemental data

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Sample components

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Entire : RNA polymerase II transcription pre-initiation complex

EntireName: RNA polymerase II transcription pre-initiation complex
Components
  • Sample: RNA polymerase II transcription pre-initiation complex
  • Protein or peptide: RNA polymerase II transcription pre-initiation complex

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Supramolecule #1000: RNA polymerase II transcription pre-initiation complex

SupramoleculeName: RNA polymerase II transcription pre-initiation complex
type: sample / ID: 1000 / Number unique components: 32
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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Macromolecule #1: RNA polymerase II transcription pre-initiation complex

MacromoleculeName: RNA polymerase II transcription pre-initiation complex
type: protein_or_peptide / ID: 1 / Name.synonym: PIC / Details: including HIS4(-81/+1) template DNA / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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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

Concentration0.3 mg/mL
BufferpH: 7.6
Details: 20mM HEPES, 5mM DTT, 2mM magnesium acetate, 30 mM potassium acetate
StainingType: NEGATIVE
Details: 20 ul of fixed sample was dialyzed for 2 hours to remove glycerol. 3 ul were transferred to a grid and flash frozen in liquid ethane with a Vitrobot.
GridDetails: Quantifoil was glow-discharged using a Harrick Plasma Cleaner
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III / Method: blot time 3 sec, waiting 10sec, force 9

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 81,000 times magnification
DateJun 25, 2010
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 15 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: frame
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SIMPLE / Number images used: 433916
DetailsPIC particles were windowed semi-automatically using the EMAN program Boxer

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