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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 (open)
Method:Single particle reconstruction (12.7 angstroms resolution)
Red flagLatest update:2013-04-03
Sample
Sample name: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE (open)
Theoretical molecular weight of the sample: 0.90
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinTATA-box-binding protein0.038trueHomo sapiens
2proteinGeneral transcription factor IIA0.054trueHomo sapiens
3proteinGeneral transcription factor IIB0.035trueHomo sapiens
4proteinGeneral transcription factor IIF0.087trueHomo sapiens
5proteinGeneral transcription factor IIE0.082trueHomo sapiens
6proteinDNA-directed RNA polymerase II0.55falseHomo sapiens
7nucleic-acidCore promoter DNA0.054trueHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.90.09 mg/mL10 mM HEPES, 5% glycerol, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.05% NP-40400-mesh C-flats with thin carbon support over 4um holes with 2um spacing (Protochips Inc.)
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100% KFEI VITROBOT MARK IIIncubation on grid for 5 minutes before blotting 4 seconds at -1 offset. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD2.2 mm1200 nm2400 nm100000100000FIELD 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
Gatan 626 liquid nitrogen cooledGATAN LIQUID NITROGEN°° eV K K K mrad20 e/Å2Data acquired using Leginon18-MAY-2012
Processing
Protocol:Projection matching
Software:EMAN2, SPARX
CTF correction:whole micrograph
Number of particles:53505
Imposed symmetry:C1
Resolution by author:12.7 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
2800 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails