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    Yorodumi
    - EMDB-1618: 3D reconstruction of heterodimeric yeast Pol alpha using electron... -

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    Basic information

    Entry
    Database: EMDB / ID: 1618
    Title3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy
    KeywordsDNA replication / DNA Polymerase alpha / Electron microscopy
    SampleYeast DNA polymerase alpha
    SourceSaccharomyces cerevisiae / yeast / Baker's yeast /
    Map data3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy
    Methodsingle particle reconstruction, at 22.9 A resolution
    AuthorsKlinge S / Nunez-Ramirez R / Llorca O / Pellegrini L
    CitationEMBO J., 2009, 28, 1978-1987

    EMBO J., 2009, 28, 1978-1987 StrPapers
    3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases.
    Sebastian Klinge / Rafael Núñez-Ramírez / Oscar Llorca / Luca Pellegrini

    DateDeposition: May 4, 2009 / Header (metadata) release: May 7, 2009 / Map release: May 6, 2011 / Last update: May 4, 2009

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    Structure visualization

    Movie
    • Surface view with section colored by density value
    • Surface level: 0.045
    • Imaged by UCSF CHIMERA
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    • Surface view colored by radius
    • Surface level: 0.045
    • Imaged by UCSF CHIMERA
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    Map

    Fileemd_1618.map.gz (map file in CCP4 format, 1025 KB)
    Projections & slicesSize of images:
    AxesZ (Sec.)Y (Row.)X (Col.)
    64 pix
    3.8 A/pix
    = 243.2 A
    64 pix
    3.8 A/pix
    = 243.2 A
    64 pix
    3.8 A/pix
    = 243.2 A

    Surface

    Projections

    Slices (1/3)

    Slices (1/2)

    Slices (2/3)

    Images are generated by Spider package.

    Voxel sizeX=Y=Z: 3.8 A
    Density
    Contour Level:0.045 (by author), 0.045 (movie #1):
    Minimum - Maximum-0.0982105 - 0.13849
    Average (Standard dev.)0.00211433 (0.0123166)
    Details

    EMDB XML:

    Space Group Number1
    Map Geometry
    Axis orderXYZ
    Dimensions646464
    Origin000
    Limit636363
    Spacing646464
    CellA=B=C: 243.2 A
    Alpha=beta=gamma: 90 deg.

    CCP4 map header:

    modeImage stored as Reals
    A/pix X/Y/Z3.83.83.8
    M x/y/z646464
    origin x/y/z-0.000-0.000-0.000
    length x/y/z243.200243.200243.200
    alpha/beta/gamma90.00090.00090.000
    start NX/NY/NZ-17-17-200
    NX/NY/NZ123123401
    MAP C/R/S123
    start NC/NR/NS000
    NC/NR/NS646464
    D min/max/mean-0.0980.1380.002

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    Supplemental data

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    Sample components

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    Entire Yeast DNA polymerase alpha

    EntireName: Yeast DNA polymerase alpha / Number of components: 2
    Oligomeric State: One monomer of DNA polymerase alpha binds to a monomer of B-subunit
    MassTheoretical: 185 kDa / Measured by: Gel Filtration

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    Component #1: protein, DNA polymerase alpha

    ProteinName: DNA polymerase alpha / a.k.a: DNA polymerase alpha / Oligomeric Details: Dimer / Number of Copies: 1 / Recombinant expression: Yes
    MassTheoretical: 130 kDa / Experimental: 130 kDa
    SourceSpecies: Saccharomyces cerevisiae / yeast / Baker's yeast /
    External referencesGene Ontology: GO: 0005658, GO: 0005739, GO: 0003677, GO: 0003887, GO: 0001882, GO: 0000166, GO: 0005515, GO: 0006270, GO: 0000731, GO: 0006273, GO: 0006279, GO: 0006278
    InterPro: InterPro: 006172, InterPro: 006134, InterPro: 017966, InterPro: 017964, InterPro: 006133, InterPro: 004578, InterPro: 015088

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    Component #2: protein, B subunit

    ProteinName: B subunit / a.k.a: B subunit / Oligomeric Details: Dimer / Recombinant expression: Yes / Number of Copies: 1
    MassTheoretical: 55 kDa / Experimental: 55 kDa
    SourceSpecies: Saccharomyces cerevisiae / yeast / Baker's yeast /
    External referencesInterPro: InterPro: 007185, InterPro: 016722, InterPro: 013627
    Gene Ontology: GO: 0005658, GO: 0005635, GO: 0003677, GO: 0003887, GO: 0005515, GO: 0006270, GO: 0006273, GO: 0016233

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    Experimental details

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    Sample preparation

    Specimen stateparticle
    Sample solutionSpecimen conc.: 0.7 mg/ml / Buffer solution: 100mM TrisHCl pH 8.0, 500 mM NaCl / pH: 8
    Support film400 mesh copper grid
    StainingA diluted solution of Pol alpha complex was adsorbed onto glow-discharged carbon coated grids, stained with 2% uranyl formate
    VitrificationInstrument: NONE / Cryogen name: NONE

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    Electron microscopy imaging

    ImagingMicroscope: JEOL 1230 / Date: Aug 20, 2009 / Details: Microscope used JEOL-1230
    Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 100 kV / Electron dose: 15 e/A2 / Illumination mode: FLOOD BEAM
    LensMagnification: 50000 X (nominal)
    Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
    Cs: 2.9 mm / Imaging mode: BRIGHT FIELD / Defocus: 750 - 1200 nm
    Specimen HolderHolder: Eucentric / Model: JEOL / Temperature: 293 K ( 293 - 293 K)
    CameraDetector: KODAK SO-163 FILM

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    Image acquisition

    Image acquisitionNumber of digital images: 170 / Scanner: OTHER / Sampling size: 10 microns / Bit depth: 8

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    Image processing

    ProcessingMethod: single particle reconstruction / Number of class averages: 190 / Number of projections: 12913 / Applied symmetry: C1 (asymmetric)
    3D reconstructionAlgorithm: ML3D / Software: EMAN, Xmipp / CTF correction: reverse phases / Resolution: 22.9 A / Resolution method: FSC 0.5

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    Atomic model buiding

    Modeling #1Software: adpem / Refinement protocol: rigid body / Target criteria: cross-correlation / Refinement space: REAL / Details: Protocol: Rigid Body
    Input PDB model: 3FLO
    Modeling #2Software: adpem / Refinement protocol: rigid body / Target criteria: cross-correlation / Refinement space: REAL / Details: Protocol: Rigid Body
    Input PDB model: 2VWJ

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