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    Yorodumi
    - EMDB-1893: EcoKI Type I DNA restriction-modification enzyme complex in close... -

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    Basic information

    Entry
    Database: EMDB / ID: 1893
    TitleEcoKI Type I DNA restriction-modification enzyme complex in closed state with bound 75bp cognate DNA fragment. 3D reconstruction by single particle analysis from negative stain EM.
    KeywordsEcoKI / endonuclease / methyltransferase / type I restriction / DNA / HsdS / HsdM / HsdR / electron microscopy / negative stain / translocase / DEAD-box / ATPase
    SampleEcoKI R2 M2 S1 complex with 75 bp cognate dsDNA fragment
    SourceEscherichia coli / bacteria /
    Synthetic construct
    Map data3D reconstruction of EcoKI Type I restriction-modification enzyme with DNA fragment, from negative stain EM data.
    Methodsingle particle reconstruction, at 20 A resolution
    AuthorsKennaway CK / Taylor JE / Song CF / Potrzebowski W / White JH / Swiderska A / Obarska-Kosinska A / Callow P / Cooper LP / Roberts GA / Bujnicki JM / Trinick J / Kneale GG / Dryden DTF
    CitationGenes Dev., 2012, 26, 92-104

    Genes Dev., 2012, 26, 92-104 StrPapers
    Structure and operation of the DNA-translocating type I DNA restriction enzymes.
    Christopher K Kennaway / James E Taylor / Chun Feng Song / Wojciech Potrzebowski / William Nicholson / John H White / Anna Swiderska / Agnieszka Obarska-Kosinska / Philip Callow / Laurie P Cooper / Gareth A Roberts / Jean-Baptiste Artero / Janusz M Bujnicki / John Trinick / G Geoff Kneale / David T F Dryden

    DateDeposition: Apr 6, 2011 / Header (metadata) release: Feb 15, 2012 / Map release: Feb 15, 2012 / Last update: Apr 6, 2011

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    Structure visualization

    Movie
    • Surface view with section colored by density value
    • Surface level: 2.1
    • Imaged by UCSF CHIMERA
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    • Surface view colored by radius
    • Surface level: 2.1
    • Imaged by UCSF CHIMERA
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    Map

    Fileemd_1893.map.gz (map file in CCP4 format, 433 KB)
    Projections & slicesSize of images:
    AxesZ (Sec.)Y (Row.)X (Col.)
    48 pix
    5.27 A/pix
    = 252.96 A
    48 pix
    5.27 A/pix
    = 252.96 A
    48 pix
    5.27 A/pix
    = 252.96 A

    Surface

    Projections

    Slices (1/3)

    Slices (1/2)

    Slices (2/3)

    Images are generated by Spider package.

    Voxel sizeX=Y=Z: 5.27 A
    Density
    Contour Level:2.1 (by author), 2.1 (movie #1):
    Minimum - Maximum-4.74770546 - 9.4093132
    Average (Standard dev.)0E-8 (1)
    Details

    EMDB XML:

    Space Group Number1
    Map Geometry
    Axis orderXYZ
    Dimensions484848
    Origin-24-24-24
    Limit232323
    Spacing484848
    CellA=B=C: 252.95999 A
    Alpha=beta=gamma: 90 deg.

    CCP4 map header:

    modeImage stored as Reals
    A/pix X/Y/Z5.275.275.27
    M x/y/z484848
    origin x/y/z0.0000.0000.000
    length x/y/z252.960252.960252.960
    alpha/beta/gamma90.00090.00090.000
    start NX/NY/NZ-56-56-55
    NX/NY/NZ112112112
    MAP C/R/S123
    start NC/NR/NS-24-24-24
    NC/NR/NS484848
    D min/max/mean-4.7489.409-0.000

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    Supplemental data

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    Sample components

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    Entire EcoKI R2 M2 S1 complex with 75 bp cognate dsDNA fragment

    EntireName: EcoKI R2 M2 S1 complex with 75 bp cognate dsDNA fragment
    Details: Stained with uranyl acetate / Number of components: 4 / Oligomeric State: 1x HsdS, 2x HsdM, 2x HsdR, 1x dsDNA
    MassTheoretical: 440 kDa / Experimental: 420 kDa / Measured by: Small angle X-ray scattering (SAXS)

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    Component #1: protein, EcoKI HsdS specificity subunit

    ProteinName: EcoKI HsdS specificity subunit / a.k.a: HsdS / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: No
    MassTheoretical: 50 kDa
    SourceSpecies: Escherichia coli / bacteria / / Strain: R
    Source (natural)Location in cell: Cytoplasm
    External referencesGene Ontology: GO: 0005515 / InterPro: InterPro: 000055

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    Component #2: protein, EcoKI HsdM methyltransferase subunit

    ProteinName: EcoKI HsdM methyltransferase subunit / a.k.a: HsdM / Oligomeric Details: Dimer / Recombinant expression: No / Number of Copies: 2
    MassTheoretical: 59 kDa
    SourceSpecies: Escherichia coli / bacteria / / Strain: R
    Source (natural)Location in cell: cytoplasm
    External referencesInterPro: InterPro: 003356 / Gene Ontology: GO: 0005515

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    Component #3: protein, EcoKI HsdR endonuclease subunit

    ProteinName: EcoKI HsdR endonuclease subunit / a.k.a: HsdR / Oligomeric Details: Monomer / Number of Copies: 2 / Recombinant expression: No
    MassTheoretical: 120 kDa
    SourceSpecies: Escherichia coli / bacteria / / Strain: R
    External referencesInterPro: InterPro: 004473 / Gene Ontology: GO: 0009307

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    Component #4: nucleic-acid, Deoxyribonucleic acid

    Nucleic-acidName: Deoxyribonucleic acid / a.k.a: DNA / Class: DNA / Structure: DOUBLE HELIX / Synthetic: Yes
    Sequence:
    AACNNNNNNG TGC
    MassTheoretical: 15 kDa
    SourceSpecies: Synthetic construct

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    Experimental details

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    Sample preparation

    Specimen stateparticle
    Sample solutionSpecimen conc.: 0.05 mg/ml / Buffer solution: 20mM Tris-Cl, 100 mM NaCl / pH: 4.7
    Support film400 mesh copper with continuous carbon
    StainingProtein was adsorbed onto UV treated carbon for 1 minute, blotted, then 1% uranyl acetate solution was applied for 1 min then blotted, three times.
    VitrificationInstrument: NONE / Cryogen name: NONE / Details: Negative stain

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    Electron microscopy imaging

    ImagingMicroscope: JEOL 1200EXII / Date: Jul 1, 2004 / Details: Customised JEOL 1200 EX microscope, low dose mode.
    Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 80 kV / Electron dose: 40 e/A2 / Illumination mode: FLOOD BEAM
    LensMagnification: 40000 X (nominal) / Astigmatism: Corrected at 80,000x / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 400 - 900 nm
    Specimen HolderHolder: Side entry / Model: JEOL / Temperature: 294 K
    CameraDetector: KODAK SO-163 FILM

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    Image acquisition

    Image acquisitionNumber of digital images: 50 / Scanner: OTHER / Sampling size: 20 microns / Bit depth: 8 / Details: Scanned on Imacon scanner

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    Image processing

    ProcessingMethod: single particle reconstruction / Number of class averages: 100 / Number of projections: 5786 / Details: The particles were manually selected using boxer. / Applied symmetry: C2 (2 fold cyclic)
    3D reconstructionAlgorithm: Consensus starting model created from a combination of methods.
    Euler angles: full range, C2 symmetry / Software: EMAN, IMAGIC, Spider, XMIPP / CTF correction: Filtered at 1st zero
    Details: Final rounds of refinement done in EMAN using selected input classes.
    Resolution: 20 A / Resolution method: FSC 0.5

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    Atomic model buiding

    Modeling #1Software: Chimera / Refinement protocol: rigid body / Target criteria: cross-correlation / Refinement space: RECIPROCAL
    Details: Protocol: rigid body. Homology model based on HsdR from EcoR124 (2W00) was fitted into the density after the core methylase (HsdS and 2x HsdM) was fitted.
    Input PDB model: 2W00
    Chain ID: 2W00_A

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