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Title:Characterization of the insertase for beta-barrel proteins of the outer mitochondrial membrane. 3-D reconstruction of the TOB complex
Authors:Klein A, Israel L, Lackey SWK, Nargang FE, Imhof A, Baumeister W, Neupert W, Thomas DR
Sample:TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
Method:Single particle reconstruction (14.7 angstroms resolution)
Red flagLatest update:2013-07-17
Sample
Sample name: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
Oligomeric state: heterotrimer with one subunit each Tob37,Tob38 and Tob55
Experimental molecular weight of the sample: 0.14
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinTob370.04860.0486monmerfalseNeurospora crassaThe TOB complex contains one copy each of T0b37, Tob38 and Tob55
2proteinTob380.03730.0373monomerfalseNeurospora crassaThe TOB complex contains one copy each of T0b37, Tob38 and Tob55
3proteinTob550.05470.0547monomerfalseNeurospora crassaThe TOB complex contains one copy each of T0b37, Tob38 and Tob55
Experiment
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.51 mg/mL1mM PMSF, 0.08%(v/v) Triton X-100, 50 mM HEPES pH 8.5lacey carbon films on 200 mesh Molybdenum grids
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE%120 KHOMEMADE PLUNGERblot for 4-5 seconds before plunging with whatman filter paper ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD2.0 mm-500 nm-3700 nm6200084270FIELD EMISSION GUNFEI EAGLE (4k x 4k) mmObjective lens astigmatism was corrected using the live FFT at imaging magnification.

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Gatan 656 side entry holderGATAN LIQUID NITROGEN eV K K95 K mrad20 e/Å211-OCT-2010
Processing
Protocol:REf. Free alignment, multi-reference alignment
Software:SPIDER, TOM_toolbox
CTF correction:Phase and astigmatism correction applied to each micrograph
Number of particles:1
Imposed symmetry:C1
Resolution by author:14.7 Å
Resolution method:FSC 0.5
Processing 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.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
546 μm/pixelImages 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.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails