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Title:Human TFIID binds to core promoter DNA in a reorganized structural state
Authors:Cianfrocco MA, Kassavetis GA, Grob P, Fang J, Juven-Gershon T, Kadonaga JT, Nogales E
Sample:holo-TFIID (canonical)
Method:Single particle reconstruction (35 angstroms resolution)
Sample
Sample name: holo-TFIID (canonical)
Theoretical molecular weight of the sample: 1.3
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinTranscription factor II-D1.3falseHomo sapiens
Experiment
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.80.2 mg/mL20 mM HEPES pH 7.8, 0.2 mM EDTA, 4 mM MgCl2, ~1% glycerol, 1 mM DTT, 3% trehalose, 50 mM KCl, 0.05% NP-40,400 mesh copper grid on C-flat support carbon with a thin carbon support over 4 um holes space 2 um apart.
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100% KFEI VITROBOT MARK IISample was incubated on grid for 30 seconds before blotting 6.5s at -1 offset. The grid was washed in 4 ul buffer to blot again for 6.5 s at -1 offset prior to plunging. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD2.2 mm1800 nm4500 nm8000010500FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mmObjective and condenser lens astigmatism was corrected at 80,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Gatan 626 liquid nitrogen cooledSIDE ENTRY, EUCENTRIC°° eV K K K mrad25 e/Å231-JAN-2012
Processing
Protocol:Common lines
Software:Sparx, EMAN2, FREALIGN
CTF correction:Per micrograph for phase flipping, per particles for refinement in FREALIGN
Number of particles:11898
Resolution by author:35 Å
Resolution method:FSC 0.143
Processing details:Particles were prepared within the APPION processing environment. The particles were automatically selected using Signature and extracted from phase flipped micrographs using values calculated by CTFFIND3. After projection matching within EMAN2/Sparx, the final map was further refined in FREALIGN.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
494 μm/pixelOTHER
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails