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Title:Structural visualization of key steps in human transcription initiation
Authors:He Y, Fang J, Taatjes DJ, Nogales E
Sample:TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
Method:Single particle reconstruction (14 angstroms resolution)
Red flagLatest update:2013-04-10
Sample
Sample name: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
Theoretical molecular weight of the sample: 1.35
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinTATA-box-binding proteintrueHomo sapiens
2proteinGeneral transcription factor IIAtrueHomo sapiens
3proteinGeneral transcription factor IIBtrueHomo sapiens
4proteinGeneral transcription factor IIFtrueHomo sapiens
5proteinGeneral transcription factor IIEtrueHomo sapiens
6proteinGeneral transcription factor IIHfalseHomo sapiens
7proteinDNA-directed RNA polymerase IIfalseHomo sapiens
8nucleic-acidCore promoter DNAtrueHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.90.06 mg/mL10 mM HEPES, 5% glycerol, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.05% NP-40Grids with adsorbed protein floated on 2% w/v uranyl formate for 1 minute.200 mesh Cu grid
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD2.2 mm500 nm1200 nm8000080000FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mmobjective lens astigmatism was corrected at 250,000 times magnification.

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Room temp single tiltSIDE ENTRY, EUCENTRIC°° eV K K K mrad20 e/Å2Data acquired using Leginon15-APR-2012
Processing
Protocol:Projection matching
Software:EMAN2, SPARX
CTF correction:whole micrograph
Number of particles:64712
Imposed symmetry:C1
Resolution by author:14 Å
Resolution method:FSC 0.143
Processing details:Image processing was performed in the Appion processing environment. 3D reconstruction was performed using EMAN2 and SPARX libraries. Final map was filtered to local resolution using the blocres function in Bsoft package.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
600 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails