EMD-2490 Experiments and Validation

Structure of the 39S Large Subunit of the Mammalian Mitochondrial Ribosome

Single particle reconstruction
Overview of EMD-2490
Sample name: 39S large subunit of the porcine mitochondrial ribosome
Organism: Sus scrofa domesticus
Fitted atomic model: 4ce4

Map parameters

Minimum density: -45.891
Maximum density: 48.929
Average density: -0.047
Standard deviation: 3.83
Recommended contour level: 11.00 (author)

Sample information

Sample name: 39S large subunit of the porcine mitochondrial ribosome
Cellular component: 39S large subunit of the mitochondrial ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Spider, IMAGIC-5, CTFFIND3, RELION
Number of particles: 232181
Reconstruction protocol: projection matching
CTF correction: per detector frame
Details: An initial reconstruction was obtained using the angular reconstitution approach in Imagic-5. For visualization purposes the final map was amplitude and MTF-corrected in RELION. Particles were selected and extracted in batchboxer (EMAN 1.9).

Fitting

Initial atomic model: 3V2D
Fitting software: Chimera, O, COOT
Fitting protocol: rigid body
Refinement space: REAL
Details: Ribosomal proteins and rRNA were docked into the density using Chimera. Individual domains were manually fitted an rebuild using COOT and O.

Imaging

Session 1
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 75000.0
Defocus max (nm): 3,300.00
Defocus min (nm): 1,500.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 2
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 3
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 4
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 5
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 6
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 7
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Session 8
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 3,600.00
Defocus min (nm): 1,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Sampling interval (μm): 14.0
Details: For data processing Falcon 2 data were resampled to 1.41 Angstrom per pixel

Specimen

Preparation
Specimen state: particle
Buffer: 20 mM HEPES-KOH, 100 mM KCl, 1 mM DTT, 20 mM MgCl2
Specimen support: 200 mesh Quantifoil R2/1 and R 2/2 holey carbon grids
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE