EMD-5799 Experiments and Validation

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

Single particle reconstruction
Overview of EMD-5799
Sample name: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
Organisms: Escherichia coli, synthetic construct, unidentified
Fitted atomic model: 4v7c
Related in-frame EM entries: EMD-5796, EMD-5797, EMD-5798, EMD-5800
Related EM entries by publication: EMD-5796, EMD-5797, EMD-5798, EMD-5800

Map parameters

Recommended contour level: 0.32 (author)
Number of grid points: 320 × 320 × 320
Voxel size: 1.04 × 1.04 × 1.04 Å
Minimum density: -0.46
Maximum density: 1.302
Average density: -0.028
Standard deviation: 0.175

Sample information

Sample name: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
Ligand: Viomycin
Nucleic acids: Transfer RNA, Messenger RNA
Ribosome: 70S ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3
Number of particles: 85115
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.

Fitting

Fitting 1
Initial atomic model: 4GD1
Fitting software: Chimera, CNS
Fitting protocol: rigid body
Target criteria: Cross-correlation
Refinement space: REAL
Details: Chain Y not used in refinement. See primary citation Supplementary Information for details.
Fitting 2
Initial atomic model: 3R8S
Fitting software: Chimera, CNS
Fitting protocol: rigid body
Target criteria: Cross-correlation
Refinement space: REAL
Details: See primary citation Supplementary Information for details.
Fitting 3
Initial atomic model: 1MMS
Fitting software: Chimera, CNS
Fitting protocol: rigid body
Target criteria: Cross-correlation
Refinement space: REAL
Details: Used to rebuild the L11 stalk.
Fitting 4
Initial atomic model: 3U4M
Fitting software: Chimera, CNS
Fitting protocol: rigid body
Target criteria: Cross-correlation
Refinement space: REAL
Details: Used to build homology model for L1.
Fitting 5
Initial atomic model: 1ZAV
Fitting software: Chimera, CNS
Fitting protocol: rigid body
Target criteria: Cross-correlation
Refinement space: REAL
Details: Used to build homology model for L10.

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.