EMD-5036 Experiments and Validation

Aminoacyl-tRNA-EF-Tu-GDP-kir ternary complex-bound E. coli 70S ribosome

Single particle reconstruction
Overview of EMD-5036
Sample name: 70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.
Organism: Escherichia coli
Fitted atomic model: 4v69

Map parameters

Recommended contour level: 90.00 (author)
Number of grid points: 309 × 309 × 309
Voxel size: 1.20 × 1.20 × 1.20 Å
Minimum density: -114.578
Maximum density: 303.004
Average density: 6.031
Standard deviation: 29.145

Sample information

Sample name: 70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.
Nucleic acids: fMet-tRNAfMet, Phe-tRNAPhe, tRNA
Protein: Elongation factor Tu
Ribosome: 70S ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Spider
Number of particles: 131599
Reconstruction protocol: weighted back projection
CTF correction: Correction of reconstruction of each defocus group

Fitting

Fitting 1
Initial atomic model: 2I2U
Fitting software: MDFF
Fitting protocol: flexible
Target criteria: RMSD, cross correlation
Refinement space: REAL
Details: Protocol: MDFF. An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
Fitting 2
Initial atomic model: 2I2V
Fitting software: MDFF
Fitting protocol: flexible
Target criteria: RMSD, cross correlation
Refinement space: REAL
Details: Protocol: MDFF. An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
Fitting 3
Initial atomic model: 1OB2
Fitting software: MDFF
Fitting protocol: flexible
Target criteria: RMSD, cross correlation
Refinement space: REAL
Details: Protocol: MDFF. An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)

Imaging

Session
Microscope model: FEI POLARA 300
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.26
Nominal magnification: 59000.0
Defocus max (nm): 4,520.00
Defocus min (nm): 1,200.00
Holder model: OTHER
Details: Low dose
Acquisition
Number of images: 304
Scanner model: ZEISS SCAI
Sampling interval (μm): 7.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.077
Buffer: 5 mM potassium phosphate, 5 mM magnesium acetate, 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8 mM putrescine, 1 mM spermidine, and 1 mM dithioerythritol
Specimen support: Thin Carbon on 300 mesh Quantifoil R2/4
Vitrification
Apparatus: FEI VITROBOT
Cryogen: ETHANE
Humidity: 90
Protocol: Blot 3 seconds before plunging
Details: Vitrification instrument: FEI vitrobot. Blot Offset at -1 mm