EMD-2491 Experiments and Validation

Recruitment Pathways in the Yeast Exosome by Electron Microscopy

Single particle reconstruction
Overview of EMD-2491
Sample name: Rrp44-Exosome incubated with RNA08
Organisms: Saccharomyces cerevisiae, synthetic construct

Map parameters

Recommended contour level: 0.04 (author)
Number of grid points: 90 × 90 × 90
Voxel size: 4.40 × 4.40 × 4.40 Å
Minimum density: -0.099
Maximum density: 0.181
Average density: -0.00
Standard deviation: 0.01

Sample information

Sample name: Rrp44-Exosome incubated with RNA08
Nucleic acid: RNA08
Proteins: Rrp44, Rrp43, Rrp4, Csl4, Rrp45, Rrp46-TAP, Rrp41, Rrp42, Mtr3, Rrp40

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SPIDER
Number of particles: 30000
Number of class averages: 600

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 30.0
Energy filter: FEI
Nominal CS (mm): 2.70
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 300

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.01
Buffer: 150mM NaCl, 50mM Tris-HCL,1mM DTT
Staining procedure: All samples were diluted at a final concentration of ~80 nM of the exosome in the non-digestive buffer and negatively stained in 2% (w/v) uranyl acetate solution following the standard deep stain procedure on holey-carbon coated EM copper grids covered with a thin layer of continuous carbon
Specimen support: 300 mesh continuous carbon
Vitrification
Apparatus: NONE
Cryogen: NONE