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Title:Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module
Authors:Tsai KL, Sato S, Tomomori-Sato C, Conaway RC, Conaway JW, Asturias FJ
Sample:Cdk8 kinase module of yeast Mediator complex
Method:Single particle reconstruction (15 angstroms resolution)
Red flagLatest update:2013-05-15
Sample
Sample name: Cdk8 kinase module of yeast Mediator complex
Experimental molecular weight of the sample: 0.43
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
1proteinMed120.1670.167falseSaccharomyces cerevisiae
2proteinMed130.1600.160falseSaccharomyces cerevisiae
3proteinCdk80.0630.063falseSaccharomyces cerevisiae
4proteinCycC0.0380.038falseSaccharomyces cerevisiae
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.60.020 mg/mL20mM HEPES, 200mM potassium acetate, 5mM beta-mercaptoethanol300 mesh Cu/Rh grids with continuous carbon, glow discharged in amylamine atmosphere
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE95%77 KHOMEMADE PLUNGER ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD1.2 mm1500 nm4000 nm6000060400FIELD EMISSION GUNKODAK SO-163 FILM mmObjective lens astigmatism was corrected at 125,000 times magnification using a CCD camera

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
GATAN LIQUID NITROGEN°° eV105 K80 K110 K mrad15 e/Å201-DEC-2011
Processing
Protocol:projection matching
Software:SPARX, SPIDER
CTF correction:Each particle
Number of particles:70000
Imposed symmetry:C1
Resolution by author:15 Å
Resolution method:FSC 0.5
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1546.35 μm/pixel16NIKON SUPER COOLSCAN 9000
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails