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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: 1698
    TitleThree-dimensional structure of TspO by electron cryo-microscopy of helical crystals.
    KeywordsTSPO / PBR / membrane protein / helical crystal
    SampleTspO and Escherichia coli polar lipid extract
    SourceRhodobacter sphaeroides / archaea
    Escherichia coli / bacteria /
    Map dataThe map contains 120 Angstrom thick slab through the tube reconstruction.
    Methodhelical reconstruction, at 10.2 A resolution
    AuthorsKorkhov VM / Sachse C / Short JM / Tate CG
    CitationStructure, 2010, 18, 677-687

    Structure, 2010, 18, 677-687 StrPapers
    Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals.
    Vladimir M Korkhov / Carsten Sachse / Judith M Short / Christopher G Tate

    DateDeposition: Feb 5, 2010 / Header (metadata) release: Jun 24, 2010 / Map release: Jun 24, 2010 / Last update: Feb 5, 2010

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    Structure visualization

    Movie
    • Surface view with section colored by density value
    • Surface level: 1.75
    • Imaged by UCSF CHIMERA
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    • Surface view colored by cylindrical radius
    • Surface level: 1.75
    • Imaged by UCSF CHIMERA
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    Map

    Fileemd_1698.map.gz (map file in CCP4 format, 24416 KB)
    Projections & slicesSize of images:
    AxesZ (Sec.)Y (Row.)X (Col.)
    100 pix
    1.2 A/pix
    = 120. A
    250 pix
    1.2 A/pix
    = 300. A
    250 pix
    1.2 A/pix
    = 300. A

    Surface

    Projections

    Slices (1/3)

    Slices (1/2)

    Slices (2/3)

    Images are generated by Spider package.

    Voxel sizeX=Y=Z: 1.2 A
    Density
    Contour Level:1.75 (by author), 1.75 (movie #1):
    Minimum - Maximum-2.11997 - 3.8827
    Average (Standard dev.)5.7552e-09 (1)
    Details

    EMDB XML:

    Space Group Number1
    Map Geometry
    Axis orderXYZ
    Dimensions250250100
    Origin-125-125-50
    Limit12412449
    Spacing250250100
    CellA: 300 A / B: 300 A / C: 120 A
    Alpha=beta=gamma: 90 deg.

    CCP4 map header:

    modeImage stored as Reals
    A/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
    alpha/beta/gamma90.00090.00090.000
    start NX/NY/NZ
    NX/NY/NZ
    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 / Number of components: 2 / Oligomeric State: Helical
    MassTheoretical: 13.5 MDa / Experimental: 13.5 MDa

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    Component #1: protein, TspO

    ProteinName: TspO / a.k.a: TspO / Oligomeric Details: Helical / Recombinant expression: Yes
    MassTheoretical: 18 kDa / Experimental: 18 kDa
    SourceSpecies: Rhodobacter sphaeroides / archaea
    Source (engineered)Expression System: Escherichia coli / bacteria / / Vector: pUI2701
    Source (natural)Location in cell: Outer membrane

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    Component #2: cellular-component, Lipid extract

    Cellular-componentName: Lipid extract / a.k.a: Lipid extract / Recombinant expression: No
    SourceSpecies: Escherichia coli / bacteria /

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    Experimental details

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    Sample preparation

    Specimen statehelical array
    Helical parametersAxial symmetry: D12 (2*12 fold dihedral) / Hand: LEFT HANDED / Delta z: 32 A / Delta phi: 9.6 deg.
    Crystal grow detailsIncubated and dialysed for 3 days with Escherichia coli polar lipid extract
    Sample solutionSpecimen conc.: 0.5 mg/ml / Buffer solution: 20 mM Tris, 100 mM NaCl, 2 mM EDTA / pH: 7.5
    VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 77.2 K / Method: Back-side blotting / Details: Vitrification instrument: Home built

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    Electron microscopy imaging

    ImagingMicroscope: FEI TECNAI F20 / Date: May 30, 2007
    Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 15 e/A2 / Illumination mode: FLOOD BEAM
    LensMagnification: 50000 X (nominal)
    Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
    Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 2163 nm
    Specimen HolderHolder: Side entry liquid nitrogen-cooled cryo specimen holder
    Model: GATAN LIQUID NITROGEN
    CameraDetector: KODAK SO-163 FILM

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    Image acquisition

    Image acquisitionNumber of digital images: 36 / Scanner: OTHER / Sampling size: 6 microns / Bit depth: 8

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    Image processing

    ProcessingMethod: helical reconstruction
    Details: 2 TspO molecules within dimer are related by an additional 2-fold axis parallel to tube axis.
    3D reconstructionAlgorithm: Iterative algebraic reconstruction
    Euler angles: 2 degree increments, 0-360 degrees around helical axis, up to 12 degrees out-of-plane tilt
    Software: SPIDER / CTF correction: Segment-specific CTF / Details: Imposition of 12-fold rotational symmetry / Resolution: 10.2 A / Resolution method: FSC 0.5

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