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- EMDB-2203: Characterization of the insertase for beta-barrel proteins of the... -

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Basic information

Entry
Database: EMDB / ID: EMD-2203
TitleCharacterization of the insertase for beta-barrel proteins of the outer mitochondrial membrane. 3-D reconstruction of the TOB complex
Map data3D reconstruction of Tob55 dimers isolated using the 9xHis tag on the Tob55 subunit. The Tob55 dimer is a complex identified in all outer membrane preparations but isolated only when the his-tag is on tob55. The dimer co-purifies with the TOB complex and was identified as a subclass of particles present in the his-Tob55 data sets. The mol. wgt. is 101 kDa. Two-fold symmetry has been applied
Sample
  • Sample: Tob55 dimer
  • Protein or peptide: Tob55 short
KeywordsTOB/SAM complex / beta-barrel proteins / Tob55/Sam50 / mitochondria outer membrane
Biological speciesNeurospora crassa (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.4 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 12, 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 cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2203.jpg

Downloads & links

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Map

FileDownload / File: emd_2203.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of Tob55 dimers isolated using the 9xHis tag on the Tob55 subunit. The Tob55 dimer is a complex identified in all outer membrane preparations but isolated only when the his-tag is on tob55. The dimer co-purifies with the TOB complex and was identified as a subclass of particles present in the his-Tob55 data sets. The mol. wgt. is 101 kDa. Two-fold symmetry has been applied
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-0.14503084 - 1.68543327
Average (Standard dev.)0.00043378 (±0.05871786)
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-0.1451.6850.000

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Supplemental data

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Sample components

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Entire : Tob55 dimer

EntireName: Tob55 dimer
Components
  • Sample: Tob55 dimer
  • Protein or peptide: Tob55 short

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Supramolecule #1000: Tob55 dimer

SupramoleculeName: Tob55 dimer / type: sample / ID: 1000
Details: The complexes were monodisperse. A mitochondrial outer membrane complex which can be identified as a component of the outer mitochondrial membrane but whose function is unknown.
Oligomeric state: dimeric / Number unique components: 1
Molecular weightExperimental: 101 KDa / Theoretical: 101 KDa
Method: Blue native gel electrophoresis and Isotope dilution mass spectroscopy analysis of bands isolated from BNGE gels.

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Macromolecule #1: Tob55 short

MacromoleculeName: Tob55 short / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: No
Source (natural)Organism: Neurospora crassa (fungus) / Strain: Tob55 short HT / Organelle: mitochondria / Location in cell: outer membrane
Molecular weightExperimental: 50.7 KDa / Theoretical: 50.7 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 / Instrument: HOMEMADE PLUNGER
Method: blot for 4-5 seconds before plunging with whatman filter paper #1

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using the live FFT at imaging magnification.
DateOct 7, 2009
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 20 e/Å2
Details: Images collected using TOM_acquisition software. 24800 dimers were identified in various his-Tob55 datasets. In the end 19500 were included in the final reconstruction. Two-fold symmetry was ...Details: Images collected using TOM_acquisition software. 24800 dimers were identified in various his-Tob55 datasets. In the end 19500 were included in the final reconstruction. Two-fold symmetry was applied during the last 5 rounds of refinement.
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 / 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: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.4 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. 24800 dimers were identified in various his-Tob55 datasets. In the end 19500 were included in the final reconstruction. Two-fold symmetry was applied during the last 5 rounds of refinement.
Number images used: 1

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