EMD-2373 Experiments and Validation

ribosome-RelA complex

Single particle reconstruction
Overview of EMD-2373
Sample name: 70S-RelA complex
Organisms: Thermus thermophilus, Escherichia coli

Map parameters

Recommended contour level: 0.175 (author)
Number of grid points: 210 × 210 × 210
Voxel size: 1.75 × 1.75 × 1.75 Å
Minimum density: -0.225
Maximum density: 1.175
Average density: 0.039
Standard deviation: 0.155

Sample information

Sample name: 70S-RelA complex
Nucleic acids: deacylated tRNA, tRNA, tRNA
Protein: RelA
Ribosome: 70S ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: XMIPP, SPIDER
Number of particles: 28791
Reconstruction protocol: projection matching
CTF correction: defocus groups

Fitting

Fitting 1
Initial atomic model: 2WRN
Fitting software: MDFF
Fitting protocol: flexible
Refinement space: REAL
Fitting 2
Initial atomic model: 2WRO
Fitting software: MDFF
Fitting protocol: flexible
Refinement space: REAL

Imaging

Session
Microscope model: JEOL 2200FS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 10.0
Energy filter: in-column Omega filter
Energy window (eV): 15.00
Nominal CS (mm): 2.00
Nominal magnification: 40000.0
Defocus max (nm): 4,000.00
Defocus min (nm): 1,000.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 425
Scanner model: OTHER
Sampling interval (μm): 7.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.25
Buffer: 5mM Hepes-KOH pH 7.45, 50mM KCl, 10mM NH4Cl, 10mM Mg-acetate, 5mM beta-mercapto-ethanol
Staining procedure: cryo
Specimen support: Quantifoil grids (2/4) with thin carbon on top
Vitrification
Apparatus: FEI VITROBOT MARK I
Cryogen: ETHANE
Humidity: 100
Protocol: Blot for 3 seconds before plunging