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- EMDB-1698: Three-dimensional structure of TspO by electron cryo-microscopy o... -

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Basic information

Entry
Database: EMDB / ID: EMD-1698
TitleThree-dimensional structure of TspO by electron cryo-microscopy of helical crystals.
Map dataThe map contains 120 Angstrom thick slab through the tube reconstruction.
Sample
  • Sample: TspO and Escherichia coli polar lipid extract
  • Organelle or cellular component: Lipid extract
  • Protein or peptide: TspO
KeywordsTSPO / PBR / membrane protein / helical crystal
Biological speciesEscherichia coli (E. coli) / Rhodobacter sphaeroides (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 10.2 Å
AuthorsKorkhov VM / Sachse C / Short JM / Tate CG
CitationJournal: Structure / Year: 2010
Title: Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals.
Authors: Vladimir M Korkhov / Carsten Sachse / Judith M Short / Christopher G Tate /
Abstract: The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The ...The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 A resolution. The structure suggests that monomeric TspO comprises five transmembrane alpha helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.
History
DepositionFeb 5, 2010-
Header (metadata) releaseJun 24, 2010-
Map releaseJun 24, 2010-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.75
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1698.tif

Downloads & links

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Map

FileDownload / File: emd_1698.map.gz / Format: CCP4 / Size: 23.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe map contains 120 Angstrom thick slab through the tube reconstruction.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 100 pix.
= 120. Å		1.2 Å/pix.
x 250 pix.
= 300. Å		1.2 Å/pix.
x 250 pix.
= 300. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.75 / Movie #1: 1.75
Minimum - Maximum-2.11997 - 3.8827
Average (Standard dev.)0.0000000057552 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-125-125-50
Dimensions250250100
Spacing250250100
CellA: 300 Å / B: 300 Å / C: 120 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z250250100
origin x/y/z0.0000.0000.000
length x/y/z300.000300.000120.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-125-125-50
NC/NR/NS250250100
D min/max/mean-2.1203.8830.000

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Supplemental data

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Sample components

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Entire : TspO and Escherichia coli polar lipid extract

EntireName: TspO and Escherichia coli polar lipid extract
Components
  • Sample: TspO and Escherichia coli polar lipid extract
  • Organelle or cellular component: Lipid extract
  • Protein or peptide: TspO

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Supramolecule #1000: TspO and Escherichia coli polar lipid extract

SupramoleculeName: TspO and Escherichia coli polar lipid extract / type: sample / ID: 1000 / Oligomeric state: Helical / Number unique components: 2
Molecular weightExperimental: 13.5 MDa / Theoretical: 13.5 MDa

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Supramolecule #1: Lipid extract

SupramoleculeName: Lipid extract / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Lipid extract / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: TspO

MacromoleculeName: TspO / type: protein_or_peptide / ID: 1 / Name.synonym: TspO / Oligomeric state: Helical / Recombinant expression: Yes
Source (natural)Organism: Rhodobacter sphaeroides (bacteria) / synonym: Rhodobacter sphaeroides / Location in cell: Outer membrane
Molecular weightExperimental: 18 KDa / Theoretical: 18 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pUI2701

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris, 100 mM NaCl, 2 mM EDTA
VitrificationCryogen name: ETHANE / Chamber temperature: 77.2 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home built / Method: Back-side blotting
DetailsIncubated and dialysed for 3 days with Escherichia coli polar lipid extract

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateMay 30, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6 µm / Number real images: 36 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.163 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Details2 TspO molecules within dimer are related by an additional 2-fold axis parallel to tube axis.
Final reconstructionApplied symmetry - Helical parameters - Δz: 32 Å
Applied symmetry - Helical parameters - Δ&Phi: 9.6 °
Applied symmetry - Helical parameters - Axial symmetry: D12 (2x12 fold dihedral)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: Imposition of 12-fold rotational symmetry
CTF correctionDetails: Segment-specific CTF
Final angle assignmentDetails: 2 degree increments, 0-360 degrees around helical axis, up to 12 degrees out-of-plane tilt

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