EMD-2173 Experiments and Validation

Negative stain electron microscopy of a CSN-SCF~Nedd8/Skp2/Cks1 complex

Single particle reconstruction
Overview of EMD-2173
Sample name: CSN-SCF~Nedd8/Skp2/Cks1
Organism: Homo sapiens
Related EM entries by publication: EMD-2176, EMD-2177, EMD-2174, EMD-2175

Map parameters

Minimum density: -14.358
Maximum density: 18.374
Average density: 0.213
Standard deviation: 2.608
Recommended contour level: 3.00 (author)

Sample information

Sample name: CSN-SCF~Nedd8/Skp2/Cks1
Proteins: Csn1, Csn2, Csn3, Csn4, Csn5, Csn6, Csn7b, Csn8, Cul1, Rbx1, Skp1, Skp2, Cks1, Nedd8

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: IMAGIC, Spider, EMAN, in, house
Number of particles: 8055
Reconstruction protocol: angular reconstitution in IMAGIC

Fitting

Initial atomic model: 1LDK
Fitting software: Chimera, Uro
Fitting protocol: rigid body
Refinement space: REAL
Details: Protocol: Rigid body

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 100.0
Nominal magnification: 50000.0
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Details: data was collected on a CCD camera

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.20
Buffer: 15 mM HEPES pH 7.8, 150 mM NaCl, 1% glycerol and 1 mM DTT
Staining procedure: Quantifoil 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
Apparatus: NONE
Cryogen: NONE