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    Yorodumi
    - EMDB-5362: New mRNA-tRNA translocation intermediates revealed by cryoEM: Cla... -

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    Basic information

    Entry
    Database: EMDB / ID: 5362
    TitleNew mRNA-tRNA translocation intermediates revealed by cryoEM: Class 6 (rotated 70S ribosome with A-site and P-site tRNAs)
    Keywordsribosome / 70S / rotated / intermediates / translocation / tRNA
    SampleTrp-tRNA-EFTu-GDP-kir-70S ribosome
    SourceEscherichia coli / bacteria /
    Map dataCryo-EM-revealed rotated 70S ribosome with A and P site tRNAs (class 6)
    Methodsingle particle reconstruction, at 11.5 A resolution
    AuthorsAgirrezabala X / Liao HY / Schreiner E / Fu J / Ortiz-Meoz RF / Schulten K / Green R / Frank J
    CitationProc. Natl. Acad. Sci. U.S.A., 2012, 109, 6094-6099

    Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 6094-6099 StrPapers
    Structural characterization of mRNA-tRNA translocation intermediates.
    Xabier Agirrezabala / Hstau Y Liao / Eduard Schreiner / Jie Fu / Rodrigo F Ortiz-Meoz / Klaus Schulten / Rachel Green / Joachim Frank

    DateDeposition: Dec 2, 2011 / Header (metadata) release: Jan 26, 2012 / Map release: Feb 16, 2012 / Last update: Dec 2, 2011

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    Structure visualization

    Movie
    • Surface view with section colored by density value
    • Surface level: 0.15
    • Imaged by UCSF CHIMERA
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    • Surface view colored by height
    • Surface level: 0.15
    • Imaged by UCSF CHIMERA
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    • Surface view with fitted model
    • Atomic models: : PDB-4v6r
    • Surface level: 0.15
    • Imaged by UCSF CHIMERA
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    Supplemental images

    Downloads & links

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    Map

    Fileemd_5362.map.gz (map file in CCP4 format, 61037 KB)
    Projections & slicesSize of images:
    AxesZ (Sec.)Y (Row.)X (Col.)
    250 pix
    1.5 A/pix
    = 375. A
    250 pix
    1.5 A/pix
    = 375. A
    250 pix
    1.5 A/pix
    = 375. A

    Surface

    Projections

    Slices (1/3)

    Slices (1/2)

    Slices (2/3)

    Images are generated by Spider package.

    Voxel sizeX=Y=Z: 1.5 A
    Density
    Contour Level:0.1 (by author), 0.15 (movie #1):
    Minimum - Maximum-0.49196333 - 1.57512212
    Average (Standard dev.)0.02891555 (0.20386297)
    Details

    EMDB XML:

    Space Group Number1
    Map Geometry
    Axis orderXYZ
    Dimensions250250250
    Origin-124-124-124
    Limit125125125
    Spacing250250250
    CellA=B=C: 375 A
    Alpha=beta=gamma: 90 deg.

    CCP4 map header:

    modeImage stored as Reals
    A/pix X/Y/Z1.51.51.5
    M x/y/z250250250
    origin x/y/z0.0000.0000.000
    length x/y/z375.000375.000375.000
    alpha/beta/gamma90.00090.00090.000
    start NX/NY/NZ-62-62-62
    NX/NY/NZ125125125
    MAP C/R/S123
    start NC/NR/NS-124-124-124
    NC/NR/NS250250250
    D min/max/mean-0.4921.5750.029

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    Supplemental data

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    Sample components

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    Entire Trp-tRNA-EFTu-GDP-kir-70S ribosome

    EntireName: Trp-tRNA-EFTu-GDP-kir-70S ribosome / Number of components: 3 / Oligomeric State: monomeric
    MassTheoretical: 2.8 MDa / Experimental: 2.8 MDa

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    Component #1: ribosome-prokaryote, 70S ribosome

    Ribosome-prokaryoteName: 70S ribosome / Prokaryote: ALL / Recombinant expression: No
    SourceSpecies: Escherichia coli / bacteria /

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    Experimental details

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    Sample preparation

    Specimen stateparticle
    Sample solutionSpecimen conc.: 0.03 mg/ml
    Buffer solution: HiFi buffer (50 mM Tris-HCl, pH 7.5, 70mM NH4Cl, 30 mM KCl, 3.5 mM MgCl2, 0.5 mM spermidine, 8mM putrescine, 2 mM DTT, 3.5 mM MgCl2)
    pH: 7.5
    Support film200 mesh
    VitrificationInstrument: FEI VITROBOT / Cryogen name: NITROGEN / Temperature: 80 K / Humidity: 90 % / Method: blot for 3 seconds / Details: Vitrification instrument: Vitrobot

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    Electron microscopy imaging

    ImagingMicroscope: FEI TECNAI F30 / Date: May 1, 2007
    Details: automated data collection system AutoEMation (CCD mag. 100000x)
    Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/A2 / Electron beam tilt params: no tilt / Illumination mode: FLOOD BEAM
    LensMagnification: 59000 X (nominal), 58269 X (calibrated)
    Astigmatism: objective corrected at 100,000 times magnification
    Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 4000 nm / Energy filter: no filter
    Specimen HolderHolder: cartridge / Model: OTHER / Temperature: 80.7 K ( 80.7 - 80.7 K)
    CameraDetector: TVIPS TEMCAM-F415 (4k x 4k)

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    Image processing

    ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 37000
    3D reconstructionSoftware: XMIPP, SPIDER / CTF correction: defocus groups, Wiener filter / Details: Classification using ML3D / Resolution: 11.5 A / Resolution method: FSC 0.5

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    Atomic model buiding

    Modeling #1Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
    Details: Protocol: Molecular Dynamics based flexible fitting
    Input PDB model: 2I2U
    Modeling #2Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
    Details: Protocol: Molecular Dynamics based flexible fitting
    Input PDB model: 2I2V
    Output model

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