EMD-1172 Experiments and Validation

Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome.

Single particle reconstruction
Overview of EMD-1172
Sample name: Ribosome complex with initiation factor 2 in GTP state
Organism: Thermus thermophilus HB8
Related EM entry by publication: EMD-1173

Map parameters

Recommended contour level: 4.20
Number of grid points: 120 × 120 × 120
Voxel size: 3.00 × 3.00 × 3.00 Å
Minimum density: -23.79
Maximum density: 30.736
Average density: 0.121
Standard deviation: 2.447

Sample information

Sample name: Ribosome complex with initiation factor 2 in GTP state
Nucleic acids: messenger RNA, formyl-methionyl-RNA
Protein: initiation factor 2
Ribosome: 70S

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: IMAGIC
Number of particles: 74300
Number of class averages: 2810
Reconstruction protocol: exact filtered back-projection
CTF correction: individual particles
Details: exact filtered back-projection The particles were selected using an semi-automatic selection procidure

Fitting

Fitting software: program O
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): 10.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 2,400.00
Defocus min (nm): 1,000.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 36
Scanner model: PRIMESCAN
Sampling interval (μm): 3.0
Details: Heidelberg Druckmaschinen

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.50
Buffer: 20mM Tris-HCl (pH7.5) ,20mM MgCl2, 100mM KCl, 1mM DTT
Staining procedure: no staining, cryo-EM with holey carbon grids
Specimen support: 300 mesh Copper/Rhodium grid
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE
Protocol: Blot for 2 seconds before plunging
Details: Vitrification instrument: home-made cryo-plunger