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- EMDB-5026: Cryo-negative stain structure of the yeast transcription factor T... -

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Basic information

Entry
Database: EMDB / ID: EMD-5026
TitleCryo-negative stain structure of the yeast transcription factor TFIID at 20 A resolution
Map dataVolume of yeast TFIID filtered to 20 A resolution
Sample
  • Sample: TAp-tag purified yeast TFIID
  • Protein or peptide: Transcription factor TFIID
KeywordsTranscription factor TFIID / cryo electron microscopy / yeast
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 22.0 Å
AuthorsPapai G / Tripathi MK / Ruhlmann C / Crucifix C / Jennings JL / Link AJ / Weil PA / Schultz P
CitationJournal: Structure / Year: 2009
Title: Mapping the initiator binding Taf2 subunit in the structure of hydrated yeast TFIID.
Authors: Gabor Papai / Manish K Tripathi / Christine Ruhlmann / Sebastiaan Werten / Corinne Crucifix / P Anthony Weil / Patrick Schultz /
Abstract: The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation ...The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules. Image analysis revealed the extent of conformational flexibility of the complex and the selection of the most homogeneous TFIID subpopulation allowed us to determine an improved structural model at 23 Angstroms resolution. This study also identified two subpopulations of Taf2p-containing and Taf2p-depleted TFIID molecules. By comparing these two TFIID species we could infer the position of Taf2p, which was confirmed by immunolabeling using a subunit-specific antibody. Mapping the position of this crucial subunit in the vicinity of Taf1p and of TBP sheds new light on its role in promoter recognition.
History
DepositionAug 20, 2008-
Header (metadata) releaseMar 11, 2009-
Map releaseMay 5, 2009-
UpdateMay 5, 2009-
Current statusMay 5, 2009Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0005
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0005
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5026_1.png

Downloads & links

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Map

FileDownload / File: emd_5026.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume of yeast TFIID filtered to 20 A resolution
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour Level1: 0.0004 / Movie #1: 0.0005
Minimum - Maximum-0.00136816 - 0.00285578
Average (Standard dev.)0.000000186638 (±0.000207027)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-79-80
Dimensions160160160
Spacing160160160
CellA=B=C: 406.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z406.400406.400406.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS-79-80-80
NC/NR/NS160160160
D min/max/mean-0.0010.0030.000

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Supplemental data

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Sample components

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Entire : TAp-tag purified yeast TFIID

EntireName: TAp-tag purified yeast TFIID
Components
  • Sample: TAp-tag purified yeast TFIID
  • Protein or peptide: Transcription factor TFIID

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Supramolecule #1000: TAp-tag purified yeast TFIID

SupramoleculeName: TAp-tag purified yeast TFIID / type: sample / ID: 1000 / Oligomeric state: monomeric / Number unique components: 1
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa

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Macromolecule #1: Transcription factor TFIID

MacromoleculeName: Transcription factor TFIID / type: protein_or_peptide / ID: 1 / Name.synonym: TFIID / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes / Database: NCBI
Source (natural)Strain: YLSTAF1 / Cell: yeast / Location in cell: nuclear protein
Molecular weightExperimental: 900 MDa / Theoretical: 900 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

Concentration1.4 mg/mL
BufferpH: 8 / Details: 10 mM Tris-HCL, 300 mM NaCl, 20% glycerol
StainingType: NEGATIVE
Details: Thin carbon film with adsorbed protein were floated on 2% w/v uranyl acetate for 30 seconds.
GridDetails: 400 mesh copper/rhodium grid with holey carbon
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: house made / Method: blot for about 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 90 K
DateSep 19, 2006
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 5.1 µm / Number real images: 121 / Average electron dose: 15 e/Å2 / Od range: 1.4 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40080 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.96 µm / Nominal defocus min: 0.137 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Side entry / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsMost homogeneous particles were sorted out by using several starting models obtained by electron tomography.
CTF correctionDetails: phase flipping on each particle from one micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, SPIDER / Number images used: 10205
Final two d classificationNumber classes: 700

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