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    Yorodumi
    - EMDB-1561: Electron tomography of isometrically contracting insect flight muscle -

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    Basic information

    Entry
    Database: EMDB / ID: 1561
    TitleElectron tomography of isometrically contracting insect flight muscle
    Keywordsinsect / muscle / myosin / troponin / tropomyosin / actin / light chains / thin filament / thick filament / electron microscopy / image processing / isometric contraction / freezing / freeze substitution / microtomy / multivariate data analysis
    SampleIsometrically contracting asynchronous insect flight muscle
    SourceLethocerus indicus / arthropod / Water bug
    Map dataThis is a dual axis tomogram of quick frozen, freeze This is a dual axis tomogram of quick frozen, freeze substituted, plastic embedded, thin sectioned insect flight muscle from Lethocerus sp.
    Methodelectron tomography
    AuthorsWu S / Liu J / Reedy MC / Tregear RT / Winkler H / Franzini-Armstrong C / Sasaki H / Lucaveche C / Goldman YE / Reedy MK / Taylor KA
    CitationPLoS ONE, 2010, 5

    primary. PLoS ONE, 2010, 5 StrPapers
    Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.
    Shenping Wu / Jun Liu / Mary C Reedy / Richard T Tregear / Hanspeter Winkler / Clara Franzini-Armstrong / Hiroyuki Sasaki / Carmen Lucaveche / Yale E Goldman / Michael K Reedy / Kenneth A Taylor

    1. J. Struct. Biol., 2009, 168, 485-502 StrPapers
    Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
    Shenping Wu / Jun Liu / Mary C Reedy / Hanspeter Winkler / Michael K Reedy / Kenneth A Taylor

    DateDeposition: Sep 26, 2008 / Header (metadata) release: Sep 29, 2008 / Map release: Apr 12, 2010 / Last update: Sep 26, 2008

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    Map

    Fileemd_1561.map.gz (map file in CCP4 format, 588001 KB)
    Projections & slicesSize of images:
    AxesZ (Sec.)Y (Row.)X (Col.)
    70 pix
    6.9 A/pix
    = 483. A
    1344 pix
    6.9 A/pix
    = 11040. A
    1600 pix
    6.9 A/pix
    = 9273.601 A

    Projections

    Slices (1/3)

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    Images are generated by Spider package.

    Voxel sizeX=Y=Z: 6.9 A
    Density
    Contour Level:
    Minimum - Maximum-3296.96 - 4848.86
    Average (Standard dev.)5.83993 (532.04)
    Details

    EMDB XML:

    Space Group Number1
    Map Geometry
    Axis orderXYZ
    Dimensions1344160070
    Origin000
    Limit1343159969
    Spacing1344160070
    CellA: 9273.6 A / B: 11040 A / C: 483 A
    Alpha=beta=gamma: 90 deg.

    CCP4 map header:

    modeImage stored as Reals
    A/pix X/Y/Z6.90000074404766.96.9
    M x/y/z1344160070
    origin x/y/z0.0000.0000.000
    length x/y/z9273.60111040.000483.000
    alpha/beta/gamma90.00090.00090.000
    start NX/NY/NZ
    NX/NY/NZ
    MAP C/R/S123
    start NC/NR/NS000
    NC/NR/NS1600134470
    D min/max/mean-3296.9594848.8645.840

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    Supplemental data

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    Sample components

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    Entire Isometrically contracting asynchronous insect flight muscle

    EntireName: Isometrically contracting asynchronous insect flight muscle
    Details: This specimen is obtained from a quick frozen, isometrically contracting asynchronous insect flight muscle that has been freeze substituted, plastic embedded, and thin sectioned
    Number of components: 8 / Oligomeric State: tissue

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    Component #1: cellular-component, myofibril

    Cellular-componentName: myofibril / a.k.a: muscle / Oligomeric Details: muscle fibril
    Details: The sample was enblock stained using uranyl acetate and tannic acid, and post stained with lead citrate and potassium permanganate
    Recombinant expression: No
    SourceSpecies: Lethocerus indicus / arthropod / Water bug
    Source (natural)Organelle: myofibril / Location in cell: myooplasm
    Organ or tissue: asynchronous dorsal longitudinal flight muscle

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    Experimental details

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    Sample preparation

    Sample solutionBuffer solution: 20 mM MOPS buffer, 5 mM NaN3, and MgCl2, ATP, CaCl2, and EGTA in varying millimolar concentrations
    StainingFreeze slammed fibers were freeze-substituted in acetone using a tannic aciduranyl acetate sequence, and ultimately embedded in Araldite-506 for thin-section electron microscopy. Ultrathin (25-30 nm) longitudinal sections were stained by permanganate-lead.
    VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: HELIUM / Temperature: 4.5 K
    Method: smash against a liquid helium cooled Au-coated Cu mirror
    Details: Vitrification instrument: modified Heuser Cryopress freezing head

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    Electron microscopy imaging

    ImagingMicroscope: FEI/PHILIPS CM300FEG/T
    Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
    LensCs: 2 mm / Imaging mode: BRIGHT FIELD
    Specimen HolderHolder: Gatan model 670 Ultrahigh tilt analytical holder / Model: OTHER / Tilt Angle: -72 - 72 deg.
    CameraDetector: TVIPS TEMCAM-F224 (2k x 2k)

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    Image acquisition

    Image acquisitionBit depth: 16
    Details: Images recorded on a TVIPS Tem-Cam F224 2k x 2k CCD camera

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    Image processing

    ProcessingMethod: electron tomography / Number of sections: 70
    3D reconstructionAlgorithm: marker free alignment / Software: PROTOMO, IMOD
    Details: Two tilt series at 90 degrees to one another were first independently aligned using marker-free alignment and area matching. The two resulting tomograms were then merged by patch correlation and volume warp using IMOD. Tomogram computed using weighted back projection.
    Resolution method: FSC 0.5

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