EMD-2167 Experiments and Validation

Cryo-EM structure of the 60S-Arx1-Rei1-Jjj1 complex

Single particle reconstruction
Overview of EMD-2167
Sample name: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1
Organism: Saccharomyces cerevisiae
Related EM entry by publication: EMD-2168, EMD-2169

Map parameters

Recommended contour level: 24.60 (author)
Number of grid points: 128 × 128 × 128
Voxel size: 3.17 × 3.17 × 3.17 Å
Minimum density: -172.467
Maximum density: 300.59
Average density: -9.784
Standard deviation: 23.989

Sample information

Sample name: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1
Proteins: Arx1, Rei1, Jjj1
Ribosome: 60S ribosomal subunit

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SPIDER
Number of particles: 8881
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 0.2.

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
Fitting 3
Initial atomic model: 2Q8K
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: correlation
Refinement space: REAL
Details: Protocol: rigid body

Imaging

Session 1
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): 4,500.00
Defocus min (nm): 1,500.00
Tilt max (degrees): 0.00
Tilt min (degrees): 0.00
Holder model: GATAN LIQUID NITROGEN
Session 2
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): 4,500.00
Defocus min (nm): 1,500.00
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 7.6, 100 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol, 0.1 mM ZnCl2
Specimen support: Quantifoil holey carbon grid R2/1
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE
Protocol: manual blotting
Details: Vitrification instrument: manual plunger