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- EMDB-2195: Characterization of the insertase for beta-barrel proteins of the... -

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Entry
Database: EMDB / ID: EMD-2195
TitleCharacterization of the insertase for beta-barrel proteins of the outer mitochondrial membrane. 3-D reconstruction of the TOB complex
Map data3D reconstruction of TOB complexes isolated using the 9xHis tag on the Tob37 subunit. The TOB complex is a hetero trimer containing one copy each of Tob37, Tob38 and Tob37. Total mol. Wgt. Is 140 kDa. In this sample there are three isoforms of Tob55 54.7, 54.1 and 50.7 kDa respectively. This affects the reconstruction.
Sample
  • Sample: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
  • Protein or peptide: Tob37
  • Protein or peptide: Tob38
  • Protein or peptide: Tob55
KeywordsTOB/SAM complex / beta-barrel proteins / Tob55/Sam50 / mitochondria outer membrane / cryo-electron microscopy
Biological speciesNeurospora crassa (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.7 Å
AuthorsKlein A / Israel L / Lackey SWK / Nargang FE / Imhof A / Baumeister W / Neupert W / Thomas DR
CitationJournal: J Cell Biol / Year: 2012
Title: Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane.
Authors: Astrid Klein / Lars Israel / Sebastian W K Lackey / Frank E Nargang / Axel Imhof / Wolfgang Baumeister / Walter Neupert / Dennis R Thomas /
Abstract: The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and ...The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55-Sam50, Tob38-Sam35, and Tob37-Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB-SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.
History
DepositionSep 11, 2012-
Header (metadata) releaseOct 10, 2012-
Map releaseNov 14, 2012-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2195.jpg

Downloads & links

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Map

FileDownload / File: emd_2195.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of TOB complexes isolated using the 9xHis tag on the Tob37 subunit. The TOB complex is a hetero trimer containing one copy each of Tob37, Tob38 and Tob37. Total mol. Wgt. Is 140 kDa. In this sample there are three isoforms of Tob55 54.7, 54.1 and 50.7 kDa respectively. This affects the reconstruction.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.78 Å/pix.
x 140 pix.
= 249.2 Å		1.78 Å/pix.
x 140 pix.
= 249.2 Å		1.78 Å/pix.
x 140 pix.
= 249.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.78 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.11285038 - 1.22826016
Average (Standard dev.)-0.00021713 (±0.06882238)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 249.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.781.781.78
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z249.200249.200249.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.1131.228-0.000

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Supplemental data

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Sample components

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Entire : TOB complex mitochondrial outer membrane complex required for ins...

EntireName: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
Components
  • Sample: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
  • Protein or peptide: Tob37
  • Protein or peptide: Tob38
  • Protein or peptide: Tob55

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Supramolecule #1000: TOB complex mitochondrial outer membrane complex required for ins...

SupramoleculeName: TOB complex mitochondrial outer membrane complex required for inserting beta-barrel proteins into the outer membrane.
type: sample / ID: 1000 / Details: The complexes were monodisperse.
Oligomeric state: heterotrimer with one subunit each Tob37,Tob38 and Tob55
Number unique components: 3
Molecular weightExperimental: 140 KDa / Theoretical: 140 KDa
Method: Blue native gel electrophoresis and Isotope dilution mass spectroscopy analysis of bands isolated from BNGE gels.

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Macromolecule #1: Tob37

MacromoleculeName: Tob37 / type: protein_or_peptide / ID: 1
Details: The TOB complex contains one copy each of T0b37, Tob38 and Tob55
Number of copies: 1 / Oligomeric state: monmer / Recombinant expression: No
Source (natural)Organism: Neurospora crassa (fungus) / Strain: Tob37HT / Organelle: mitochondria / Location in cell: outer membrane
Molecular weightExperimental: 48.6 KDa / Theoretical: 48.6 KDa

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Macromolecule #2: Tob38

MacromoleculeName: Tob38 / type: protein_or_peptide / ID: 2
Details: The TOB complex contains one copy each of T0b37, Tob38 and Tob55
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Neurospora crassa (fungus) / Strain: Tob37HT / Organelle: mitochondria / Location in cell: outer membrane
Molecular weightExperimental: 37.3 KDa / Theoretical: 37.3 KDa

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Macromolecule #3: Tob55

MacromoleculeName: Tob55 / type: protein_or_peptide / ID: 3
Details: The TOB complex contains one copy each of T0b37, Tob38 and Tob55
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Neurospora crassa (fungus) / Strain: Tob37HT / Organelle: mitochondria / Location in cell: outer membrane
Molecular weightExperimental: 54.7 KDa / Theoretical: 54.7 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction

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Sample preparation

Concentration1 mg/mL
BufferpH: 8.5
Details: 1mM PMSF, 0.08%(v/v) Triton X-100, 50 mM HEPES pH 8.5
GridDetails: lacey carbon films on 200 mesh Molybdenum grids
VitrificationCryogen name: ETHANE / Chamber temperature: 120 K / Instrument: HOMEMADE PLUNGER
Method: blot for 4-5 seconds before plunging with whatman filter paper

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMax: 95 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using the live FFT at imaging magnification.
DateOct 11, 2010
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 546 / Average electron dose: 20 e/Å2
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.
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 84270 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: -3.7 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Gatan 656 side entry holder / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase and astigmatism correction applied to each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, TOM_toolbox
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 ...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.
Number images used: 1

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