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Title:Structural visualization of key steps in human transcription initiation
Authors:He Y, Fang J, Taatjes DJ, Nogales E
Sample:apo TFIIH
Method:Single particle reconstruction (20 angstroms resolution)
Red flagLatest update:2013-04-10
Sample
Sample name: apo TFIIH
Theoretical molecular weight of the sample: 0.45
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinGeneral transcription factor IIHfalseHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.90.04 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 Leginon17-APR-2012
Processing
Protocol:Projection matching
Software:EMAN2, SPARX
CTF correction:whole micrograph
Number of particles:13023
Imposed symmetry:C1
Resolution by author:20 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
450 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails