EMD-2277 Experiments and Validation

Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles

Single particle reconstruction
Overview of EMD-2277
Sample name: T. thermophilus 70S ribosome
Organism: Thermus thermophilus
Related EM entry by publication: EMD-2275, EMD-2276

Map parameters

Minimum density: -0.58
Maximum density: 1.04
Average density: 0
Standard deviation: 0.07
Recommended contour level: 0.24 (author)

Sample information

Sample name: T. thermophilus 70S ribosome
Ribosome: 70S ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: CTFFIND3, RELION
Number of particles: 35404
CTF correction: each image
Details: Use a newly developed statistical movie processing approach to compensate for beam-induced movement Use a newly developed statistical movie processing to compensate for beam-induced movement

Fitting

Fitting 1
Initial atomic model: 2WH1
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Details: Protocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
Fitting 2
Initial atomic model: 2WH2
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Details: Protocol: Rigid body. The domains were separately fitted by manual docking using program Chimera

Imaging

Session
Microscope model: FEI POLARA 300
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 16.0
Nominal CS (mm): 2
Nominal magnification: 59000.0
Defocus max (nm): 3,597
Defocus min (nm): 1,178
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 360
Sampling interval (μm): 14.0
Details: Every image is the average of sixteen frames recorded by the direct electron detector

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.2
Buffer: 3mM Hepes-KOH, 6.6 mM Tris-acetate pH 7.2, 3 mM NH4Cl, 6.6 mM NH4-acetate, 48 mM K-acetate, 4 mM Mg-acetate, 2.4 mM DTT
Specimen support: Quantifoil grids (2/2) with 3 nm thin carbon on top
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: Blot 2.5 seconds before plunging