EMD-2168 Experiments and Validation

Cryo-EM structure of the 60S-Rei1 complex

Single particle reconstruction
Overview of EMD-2168
Sample name: 60S ribosomal subunit in complex with Rei1
Organism: Saccharomyces cerevisiae
Related EM entry by publication: EMD-2167, EMD-2169

Map parameters

Minimum density: -168.914
Maximum density: 317.857
Average density: -10.618
Standard deviation: 24.744
Recommended contour level: 27.00 (author)

Sample information

Sample name: 60S ribosomal subunit in complex with Rei1
Protein: Rei1
Ribosome: 60S ribosomal subunit

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SPIDER
Number of particles: 17415
Reconstruction protocol: projection matching
CTF correction: per frame
Details: Fourier amplitudes of the reconstruction were enhanced using the SAXS curve from ribosomes; subsequently, the map was filtered in SPIDER using a butterworth low-pass filter with a filter midpoint of 0.22.

Fitting

Fitting 1
Initial atomic model: 3U5H
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: correlation
Refinement space: REAL
Details: Protocol: rigid body
Fitting 2
Initial atomic model: 3U5I
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: correlation
Refinement space: REAL
Details: Protocol: rigid body

Imaging

Session
Microscope model: FEI TECNAI 20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.30
Nominal magnification: 83000.0
Defocus max (nm): 6,000.00
Defocus min (nm): 1,300.00
Tilt max (degrees): 0.00
Tilt min (degrees): 0.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Sampling interval (μm): 15.0
Details: images were acquired using a 2 x 2 frame spot scan (per hole) using a serial EM script

Specimen

Preparation
Specimen state: particle
Buffer: 20 mM Hepes-NaOH pH 8.3, 50 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol
Staining procedure: cryo
Specimen support: Quantifoil holey carbon grid R2/1
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE
Protocol: manual blotting
Details: Vitrification instrument: manual plunger