EMD-5797 Experiments and Validation

Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State

Single particle reconstruction
Overview of EMD-5797
Sample name: Post-translocation ribosome bound to EF-G with P, E sites occupied by tRNA
Organisms: Escherichia coli, synthetic construct, unidentified
Related in-frame EM entries: EMD-5796, EMD-5798, EMD-5799, EMD-5800
Related EM entries by publication: EMD-5796, EMD-5800, EMD-5799, EMD-5798

Map parameters

Minimum density: -0.719
Maximum density: 1.933
Average density: -0.029
Standard deviation: 0.185
Recommended contour level: 0.40 (author)

Sample information

Sample name: Post-translocation ribosome bound to EF-G with P, E sites occupied by tRNA
Ligands: Fusidic Acid, Viomycin
Nucleic acids: Transfer RNA, Messenger RNA
Protein: Elongation Factor G
Ribosome: 70S ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3
Number of particles: 1341961
Reconstruction protocol: Projection Matching
CTF correction: CTFFIND3, FREALIGN per micrograph
Details: Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details. Refinement and 3D classification performed by Frealign. See primary citation Supplementary Information for details.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 30.0
Nominal CS (mm): 0.01
Nominal magnification: 133333.0
Defocus max (nm): 6,950.00
Defocus min (nm): 1,150.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 13341
Sampling interval (μm): 14.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.40
Buffer: 10 mM HEPES-KOH, 5 mM MgCl2, 90 mM NH4Cl, 2 mM spermidine, 0.1 mM spermine, 6 mM BME, 0.5 mM viomycin, 0.5 mM GTP, 0.5 mM fusidic acid
Specimen support: C-flat 1.2/1.3 holey carbon 400 mesh copper grid, glow discharged with a current of -20 mA for 45 seconds in an EMITECH K100X glow discharge unit
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 95
Protocol: Freshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side port, blotted for 7 seconds with a positional offset of 2, and plunged into liquid ethane.