EMD-2496 Experiments and Validation

Substrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy

Single particle reconstruction
Overview of EMD-2496
Sample name: Rrp44-Exosome incubated with RNA24
Organisms: Saccharomyces cerevisiae, synthetic construct

Map parameters

Minimum density: -0.1
Maximum density: 0.19
Average density: 0
Standard deviation: 0.01
Recommended contour level: 0.05 (emdb)

Sample information

Sample name: Rrp44-Exosome incubated with RNA24
Nucleic acid: RNA24
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: 28100

Fitting

Initial atomic model: 4IFD
Used chains: A, B, C, D, E, F, J, H, I, J
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 30.0
Holder model: OTHER
Acquisition
Number of images: 300

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 1
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 continus carbon
Vitrification
Apparatus: NONE
Cryogen: NONE