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    - EMDB-1721: Ribosome dynamics and tRNA movement as visualized by time-resolve... -

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    Basic information

    Entry
    Database: EMDB / ID: 1721
    TitleRibosome dynamics and tRNA movement as visualized by time-resolved electron cryomicroscopy
    KeywordsRibosome / translation / translocation / tRNA
    SampleSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in classic post-translocation state (post1)
    SourceEscherichia coli / bacteria /
    Synthetic construct
    Map dataSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in classic post-translocation state (post1)
    Methodsingle particle reconstruction, at 12 A resolution
    AuthorsFischer N / Konevega AL / Wintermeyer W / Rodnina MV / Stark H
    CitationNature, 2010, 466, 329-333

    Nature, 2010, 466, 329-333 StrPapers
    Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.
    Niels Fischer / Andrey L Konevega / Wolfgang Wintermeyer / Marina V Rodnina / Holger Stark

    DateDeposition: Apr 30, 2010 / Header (metadata) release: Jun 16, 2010 / Map release: May 6, 2011 / Last update: Apr 30, 2010

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    Structure visualization

    Movie
    • Surface view with section colored by density value
    • Surface level: 27
    • Imaged by UCSF CHIMERA
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    • Surface view colored by height
    • Surface level: 27
    • Imaged by UCSF CHIMERA
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    • Surface view with fitted model
    • Atomic models: : PDB-4v75
    • Surface level: 27
    • Imaged by UCSF CHIMERA
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    Supplemental images

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    Map

    Fileemd_1721.map.gz (map file in CCP4 format, 8193 KB)
    Projections & slicesSize of images:
    AxesZ (Sec.)Y (Row.)X (Col.)
    128 pix
    2.8 A/pix
    = 358.4 A
    128 pix
    2.8 A/pix
    = 358.4 A
    128 pix
    2.8 A/pix
    = 358.4 A

    Surface

    Projections

    Slices (1/3)

    Slices (1/2)

    Slices (2/3)

    Images are generated by Spider package.

    Voxel sizeX=Y=Z: 2.8 A
    Density
    Contour Level:27 (by author), 27 (movie #1):
    Minimum - Maximum-122.001 - 202.135
    Average (Standard dev.)-1.09276 (20.6321)
    Details

    EMDB XML:

    Space Group Number1
    Map Geometry
    Axis orderXYZ
    Dimensions128128128
    Origin000
    Limit127127127
    Spacing128128128
    CellA=B=C: 358.4 A
    Alpha=beta=gamma: 90 deg.

    CCP4 map header:

    modeImage stored as Reals
    A/pix X/Y/Z2.82.82.8
    M x/y/z128128128
    origin x/y/z0.0000.0000.000
    length x/y/z358.400358.400358.400
    alpha/beta/gamma90.00090.00090.000
    start NX/NY/NZ-64-64-64
    NX/NY/NZ128128128
    MAP C/R/S123
    start NC/NR/NS000
    NC/NR/NS128128128
    D min/max/mean-122.001202.135-1.093

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    Supplemental data

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    Sample components

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    Entire Substate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in class...

    EntireName: Substate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in classic post-translocation state (post1)
    Number of components: 4
    MassTheoretical: 2.5 MDa

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    Component #1: ribosome-prokaryote, Ribosome

    Ribosome-prokaryoteName: Ribosome / a.k.a: E. coli 70S / Prokaryote: ALL / Recombinant expression: No
    MassTheoretical: 2.5 MDa
    SourceSpecies: Escherichia coli / bacteria /

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    Component #2: nucleic-acid, fMetVal-tRNAVal

    Nucleic-acidName: fMetVal-tRNAVal / a.k.a: peptidyl tRNA / Class: T-RNA / Structure: DOUBLE HELIX / Synthetic: No
    MassTheoretical: 25 kDa
    SourceSpecies: Escherichia coli / bacteria /

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    Component #3: nucleic-acid, tRNAfMet

    Nucleic-acidName: tRNAfMet / a.k.a: deacylated tRNA / Class: T-RNA / Structure: DOUBLE HELIX / Synthetic: No
    MassTheoretical: 25 kDa
    SourceSpecies: Escherichia coli / bacteria /

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    Component #4: nucleic-acid, m022 mRNA

    Nucleic-acidName: m022 mRNA / a.k.a: mRNA / Class: RNA / Details: Coding sequence AUGGUU / Structure: SINGLE STRANDED / Synthetic: Yes
    SourceSpecies: Synthetic construct

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    Experimental details

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    Sample preparation

    Specimen stateparticle
    Sample solutionBuffer solution: 50 mM Tris-HCl, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, 0.6 mM spermine, 0.4 mM spermidine
    pH: 7.5
    VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 77 K / Humidity: 75 % / Method: Manual blotting for about 2 seconds
    Time resolved state: Samples were vitrified at different time points along the reaction coordinate (1, 2, 5 and 20 minutes after addition of deacylated tRNAfMet to 70S-fMetVal-tRNAVal complexes)
    Details: Vitrification instrument: Custom-built CEVS. Dew-point temperature (temperature on the grid) adjusted to 18 degrees C

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    Electron microscopy imaging

    ImagingMicroscope: FEI/PHILIPS CM200FEG
    Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 160 kV / Electron dose: 20 e/A2 / Illumination mode: SPOT SCAN
    LensMagnification: 161000 X (nominal), 162740 X (calibrated)
    Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
    Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 500 - 2000 nm
    Specimen HolderHolder: Eucentric / Model: GATAN LIQUID NITROGEN / Temperature: 77 K
    CameraDetector: GENERIC TVIPS (4k x 4k)

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    Image processing

    ProcessingMethod: single particle reconstruction / Number of projections: 13207 / Applied symmetry: C1 (asymmetric)
    3D reconstructionAlgorithm: Projection matching / Software: IMAGIC, custom, Spider / CTF correction: Local
    Details: Final maps were calculated from 13 datasets acquired at different time points, computationally sorted into distinct substates
    Resolution: 12 A / Resolution method: FSC 0.5

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    Atomic model buiding

    Output model

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