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Title:Negative stain electron microscopy of a CSN-SCF~Nedd8/Skp2/Cks1 complex
Authors:Enchev RI, Scott DC, da Fonseca P, Schreiber A, Monda JK, Schulman BA, Peter M, Morris EP
Sample:CSN-SCF~Nedd8/Skp2/Cks1
Method:Single particle reconstruction (25 angstroms resolution)
Sample
Sample name: CSN-SCF~Nedd8/Skp2/Cks1
Oligomeric state: one CSN monomer and one SCF~Nedd8/Skp2/Cks1 monomer
Theoretical molecular weight of the sample: 0.5
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinCsn1monomertrueHomo sapiens
2proteinCsn2monomertrueHomo sapiens
3proteinCsn3monomertrueHomo sapiensN-terminal StrepII2x tag
4proteinCsn4monomertrueHomo sapiens
5proteinCsn5monomertrueHomo sapiensN-terminal His6 tag
6proteinCsn6monomertrueHomo sapiens
7proteinCsn7bmonomertrueHomo sapiens
8proteinCsn8monomertrueHomo sapiens
9proteinCul1monomertrueHomo sapiensN-terminal StrepII2x tag
10proteinRbx1monomertrueHomo sapiens
11proteinSkp1monomertrueHomo sapiens
12proteinSkp2monomertrueHomo sapiens
13proteinCks1monomertrueHomo sapiens
14proteinNedd8monomertrueHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.80.2 mg/mL15 mM HEPES pH 7.8, 150 mM NaCl, 1% glycerol and 1 mM DTTQuantifoil grids (R2/2 Cu 400 mesh) coated with thin carbon floated from mica were glow-discharged for 30 seconds at 50 mA and 0.2 mbar vacuum. 3 ul purified samples were applied for 1 min to the grids. Following two brief buffer washes, the grids were stained with 2% uranyl acetate, gently blotted using filter paper and air-dried.
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 F20200 kVFLOOD BEAMBRIGHT FIELD mm nm nm50000FIELD EMISSION GUNGENERIC TVIPS (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
SIDE ENTRY, EUCENTRIC°° eV K K K mrad100 e/Å210-JAN-2011
Processing
Protocol:angular reconstitution in IMAGIC
Software:IMAGIC, Spider, EMAN, in, house
Number of particles:8055
Resolution by author:25 Å
Resolution method:FSC 0.5
Processing details:See publication.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
μm/pixeldata was collected on a CCD camera
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1LDK rigid bodyChimera, UroREALProtocol: Rigid body