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Title:human RNA polymerase II in complex with B2 81-131 RNA
Authors:Kassube SA, Fang J, Grob P, Yakovchuk P, Goodrich JA, Nogales E
Sample:human RNA polymerase II in complex with B2 81-131 RNA
Method:Single particle reconstruction (25 angstroms resolution)
Red flagLatest update:2013-09-18
Sample
Sample name: human RNA polymerase II in complex with B2 81-131 RNA
Oligomeric state: RNA polymerase II (12 polypeptide chains) binds to B2 81-131 RNA
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
2nucleic-acidB2 81-131 RNA0.0162falseMus musculus
3proteinRpb110.01falseHomo sapiens
4proteinRpb100.01falseHomo sapiens
5proteinRpb90.01falseHomo sapiens
6proteinRpb80.02falseHomo sapiens
7proteinRpb70.02falseHomo sapiens
8proteinRpb60.01falseHomo sapiens
9proteinRpb50.02falseHomo sapiens
10proteinRpb40.02falseHomo sapiens
11proteinRpb30.03falseHomo sapiens
12proteinRpb20.13falseHomo sapiens
13proteinRpb10.22falseHomo 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 copper grids covered with a holey carbon film, 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 20200 kVFLOOD BEAMBRIGHT FIELD2.2 mm2000 nm5000 nm5000050280FIELD EMISSION GUNKODAK SO-163 FILM 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/Å218-SEP-2009
Processing
Protocol:projection matching
Software:EMAN2, FREALIGN
CTF correction:whole micrograph
Number of particles:31423
Resolution by author:25 Å
Resolution method:FSC 0.5
Processing details:Projection matching using a low-pass filtered model of apo Pol II as the starting model.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
25612.7 μm/pixel1.014NIKON SUPER COOLSCAN 9000
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails