EMD-2501 Experiments and Validation

Substrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy

Single particle reconstruction
Overview of EMD-2501
Sample name: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE
Organisms: Saccharomyces cerevisiae, synthetic construct

Map parameters

Recommended contour level: 0.05 (author)
Number of grid points: 120 × 120 × 120
Voxel size: 3.00 × 3.00 × 3.00 Å
Minimum density: -0.061
Maximum density: 0.131
Average density: 0.001
Standard deviation: 0.009

Sample information

Sample name: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE
Nucleic acid: methionine tRNA
Proteins: Rrp44, Rrp43, Rrp4, Csl4, Rrp45, Rrp46-TAP, Rrp41, Rrp42, Mtr3, Rrp40

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: eman, imagic, relion
Number of particles: 28800
CTF correction: both each particle and micrograph

Fitting

Initial atomic model: 4NTA
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): -3,500.00
Defocus min (nm): -500.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 1200

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 2.00
Buffer: 150mM NaCl, 50mM Tris-HCL,1mM DTT, 2mM MgCl2
Specimen support: Quantifoil grids (2/2) with2~3 nm thin carbon on top
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100
Protocol: 4 ul of the reaction solution was then applied to glow-discharged C-flat grids (1.2/1.3) covered with a layer of continuous carbon with a thickness of ~4nm. The grids were then blotted and plunged into liquid ethane in a FEI Vitrobot Mark IV