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Title:human RNA Polymerase II
Authors:Kassube SA, Fang J, Grob P, Yakovchuk P, Goodrich JA, Nogales E
Sample:human Pol II
Method:Single particle reconstruction (25 angstroms resolution)
Red flagLatest update:2013-09-18
Sample
Sample name: human Pol II
Oligomeric state: 12 subunit complex
Theoretical molecular weight of the sample: 0.52
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
1proteinRpb120.01falseHomo sapiens
2proteinRpb110.01falseHomo sapiens
3proteinRpb100.01falseHomo sapiens
4proteinRpb90.01falseHomo sapiens
5proteinRpb80.02falseHomo sapiens
6proteinRpb70.02falseHomo sapiens
7proteinRpb60.01falseHomo sapiens
8proteinRpb50.02falseHomo sapiens
9proteinRpb40.02falseHomo sapiens
10proteinRpb30.03falseHomo sapiens
11proteinRpb20.13falseHomo sapiens
12proteinRpb10.22falseHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.90.0312 mg/mL10 mM Tris pH 7.9, 10 mM HEPES pH 8.0, 4 mM MgCl2, 50 mM KCl, 0.05% NP-40, 1 mM DTT, 0.1% trehalose400 mesh C-flat grids (Protochips Inc.) with a thin carbon film floated on top
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100%86 KFEI VITROBOT MARK II6 sec blot, offset -2 ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI 20120 kVFLOOD BEAMBRIGHT FIELD2.2 mm1200 nm3000 nm80000FIELD EMISSION GUNGENERIC GATAN (4k x 4k) mmObjective lens astigmatism was corrected at 100,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Side-entry cryostageGATAN LIQUID NITROGEN°° eV78 K K K mrad20 e/Å210-JUL-2011
Processing
Protocol:projection matching
Software:EMAN2, FREALIGN
CTF correction:whole micrograph
Number of particles:8905
Resolution by author:25 Å
Resolution method:FSC 0.5
Processing details:The data set was sorted into three different conformations using an automated multi-model approach; the initial reconstructions were used as starting models for multi-model projection matching in EMAN2, followed by final refinement in FREALIGN.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
9721.5 μm/pixeldata collected with leginon
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails