EMD-2239 Experiments and Validation

Cryo-electron microscopy structure of the Trypanosoma brucei 80S ribosome

Single particle reconstruction
Overview of EMD-2239
Sample name: Trypanosoma Brucei 80S Ribosome
Organism: Trypanosoma brucei
Fitted atomic model: 4v8m

Map parameters

Minimum density: -202,702.70
Maximum density: 483,783.78
Average density: 7,679.429
Standard deviation: 33,729.66
Recommended contour level: 108,000.00 (author)

Sample information

Sample name: Trypanosoma Brucei 80S Ribosome
Ribosome: 80S Ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Spider
Number of particles: 164000
Reconstruction protocol: conjugate gradients with regularization
CTF correction: Phase-flip on each particle

Fitting

Fitting 1
Initial atomic model: 3U5B
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: cross correlation
Refinement space: REAL
Details: Protocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
Fitting 2
Initial atomic model: 2XZM
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: cross correlation
Refinement space: REAL
Details: Protocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
Fitting 3
Initial atomic model: 3IZR
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: cross correlation
Refinement space: REAL
Details: Protocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.

Imaging

Session
Microscope model: FEI POLARA 300
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 25.0
Nominal CS (mm): 2.26
Nominal magnification: 59000.0
Defocus max (nm): 4,000.00
Defocus min (nm): 1,500.00
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 1000
Scanner model: NIKON SUPER COOLSCAN 9000

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.105
Buffer: 20 mM Tris pH 7.2, 100mM MgCl2, 500 mM KCl, 5 mM beta-mercaptoethanol
Specimen support: 300 mesh Copper/Molbydenum holey carbon-coated Quantifoil 2/4 grid (Quantifoil Micro Tools GmbH) containing an additional continuous thin layer of carbon
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100
Protocol: Wait 30 sec, Blot 6 seconds, plunge