EMD-2195 Experiments and Validation

Characterization of the insertase for beta-barrel proteins of the outer mitochondrial membrane. 3-D reconstruction of the TOB complex

Single particle reconstruction
Overview of EMD-2195
Sample name: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
Organism: Neurospora crassa
Related EM entries by publication: EMD-2196, EMD-2197, EMD-2200, EMD-2201, EMD-2202, EMD-2203

Map parameters

Recommended contour level: 0.08 (author)
Number of grid points: 140 × 140 × 140
Voxel size: 1.78 × 1.78 × 1.78 Å
Minimum density: -0.113
Maximum density: 1.228
Average density: -0.00
Standard deviation: 0.069

Sample information

Sample name: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
Proteins: Tob37, Tob38, Tob55

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SPIDER, TOM_toolbox
Number of particles: 1
Reconstruction protocol: REf. Free alignment, multi-reference alignment
CTF correction: Phase and astigmatism correction applied to each micrograph
Details: Final maps were reconstructed from images that had stable alignment parameters over the last 4 rounds of refinement. Stable was defined by absolute accumulated changes in theta and psi of the projection matched of less than 10 degrees.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.00
Nominal magnification: 62000.0
Defocus max (nm): -3,700.00
Defocus min (nm): -500.00
Tilt max (degrees): 0.00
Tilt min (degrees): 0.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 546
Details: Images collected using TOM_acquisition software. 546 good micrographs were CTF corrected for phase. 175,000 particles were automatically selected. In the end 76700 were included in the final reconstruction.

Specimen

Preparation
Specimen concentration (mg/mL): 1.00
Buffer: 1mM PMSF, 0.08%(v/v) Triton X-100, 50 mM HEPES pH 8.5
Specimen support: lacey carbon films on 200 mesh Molybdenum grids
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE
Protocol: blot for 4-5 seconds before plunging with whatman filter paper