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    - PDB-3j0k: Orientation of RNA polymerase II within the human VP16-Mediator-p... -

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    Basic information

    Entry
    Database: PDB / ID: 3j0k
    TitleOrientation of RNA polymerase II within the human VP16-Mediator-pol II-TFIIF assembly
    DescriptorDNA-directed RNA polymerase II largest subunit
    DNA-directed RNA polymerase II 140 kDa polypeptide
    DNA-directed RNA polymerase II 45 kDa polypeptide
    DNA-directed RNA polymerase II 32 kDa polypeptide
    DNA-directed RNA polymerases I/II/III 27 kDa polypeptide
    and more 7 items
    KeywordsTRANSFERASE/TRANSCRIPTION / TRANSFERASE-TRANSCRIPTION complex
    Specimen sourceHomo sapiens / human
    MethodElectron microscopy (36 A resolution / Single particle / Vitreous ice (cryo EM))
    AuthorsBernecky, C. / Grob, P. / Ebmeier, C.C. / Nogales, E. / Taatjes, D.J.
    CitationPLoS Biol., 2011, 9, e1000603-e1000603

    PLoS Biol., 2011, 9, e1000603-e1000603 StrPapers
    Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.
    Carrie Bernecky / Patricia Grob / Christopher C Ebmeier / Eva Nogales / Dylan J Taatjes

    DateDeposition: Oct 4, 2011 / Release: Oct 19, 2011 / Last modification: Oct 26, 2011

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    Assembly

    Deposited unit
    A: DNA-directed RNA polymerase II largest subunit
    B: DNA-directed RNA polymerase II 140 kDa polypeptide
    C: DNA-directed RNA polymerase II 45 kDa polypeptide
    D: DNA-directed RNA polymerase II 32 kDa polypeptide
    E: DNA-directed RNA polymerases I, II, and III 27 kDa polypeptide
    F: DNA-directed RNA polymerases I, II, and III 23 kDa polypeptide
    G: DNA-directed RNA polymerase II 19 kDa polypeptide
    H: DNA-directed RNA polymerases I, II, and III 14.5 kDa polypeptide
    I: DNA-directed RNA polymerase II subunit 9
    J: DNA-directed RNA polymerases I/II/III subunit 10
    K: DNA-directed RNA polymerase II 13.6 kDa polypeptide
    L: DNA-directed RNA polymerases I, II, and III 7.7 kDa polypeptide
    hetero molecules

    470 kDa, 22 molecules
    Theoretical massNumber of molelcules
    Total
    (without water)
    470,20822
    Polyers469,59512
    Non-polymers61310
    Water0

    Omokage search
    #1idetical with deposited unit / defined by author / Symmetry operations: (identity)x1
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    Components

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    Polypeptide(L) , 10 types, 10 molecules ABCDEFGHKL

    #1polypeptide(L) / DNA-directed RNA polymerase II largest subunit / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #2polypeptide(L) / DNA-directed RNA polymerase II 140 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #3polypeptide(L) / DNA-directed RNA polymerase II 45 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #4polypeptide(L) / DNA-directed RNA polymerase II 32 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #5polypeptide(L) / DNA-directed RNA polymerases I, II, and III 27 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #6polypeptide(L) / DNA-directed RNA polymerases I, II, and III 23 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #7polypeptide(L) / DNA-directed RNA polymerase II 19 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #8polypeptide(L) / DNA-directed RNA polymerases I, II, and III 14.5 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #11polypeptide(L) / DNA-directed RNA polymerase II 13.6 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #12polypeptide(L) / DNA-directed RNA polymerases I, II, and III 7.7 kDa polypeptide / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6

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    DNA-directed RNA polymerase II subunit ... / DNA-directed RNA polymerases I/II/III subunit ... , 2 types, 2 molecules IJ

    #10polypeptide(L) / DNA-directed RNA polymerases I/II/III subunit 10 / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6
    #9polypeptide(L) / DNA-directed RNA polymerase II subunit 9 / Source: Homo sapiens (gene. exp.) / References: EC: 2.7.7.6

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    Non-polymers , 2 types, 10 molecules

    #13ChemComp-MG / MAGNESIUM ION
    #14ChemComp-ZN / ZINC ION

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    Experimental details

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    Experiment

    ExperimentMethod: ELECTRON MICROSCOPY
    EM experimentReconstruction method: SINGLE PARTICLE / Specimen type: VITREOUS ICE (CRYO EM)

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    Sample preparation

    Assembly of specimenAggregation state: PARTICLE / Composition: one Mediator complex binds one pol II-TFIIF / Mol wt theo: 1.9 MDa / Number of components: 3
    Component

    Assembly id: 1 / Ebi cell: HeLa / Ebi organelle: Nucleus / Ebi organism scientific: Homo sapiens / Type: PROTEIN / Ebi expression system: Escherichia coli

    NameIDEbi organism commonMutant flagOligomeric detailsSynonym
    core Mediator1humanNO26 subunit complexMediator
    RNA polymerase II2HumanNO12-subunit complexpol II
    TFIIF3HumanYESDimerTFIIF
    Sample preparationDetails: 20 mM HEPES, 0.10 mM EDTA, 150 mM KCl, 0.02% NP-40, 35% glycerol
    pH: 7.9
    Specimen supportDetails: thin carbon-coated holey carbon 400 mesh copper grid
    VitrificationCryogen name: ETHANE / Temp: 90 K
    Method: blot for 2 seconds, dry for 3 seconds before plunging

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    Electron microscopy imaging

    EM imagingCamera length: 0 mm
    MicroscopyMicroscope model: FEI TECNAI F20
    Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 15 e/A2 / Illumination mode: FLOOD BEAM
    Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
    Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Specimen holder type: side entry / Tilt angle max: 0 deg. / Tilt angle min: 0 deg.
    CameraType: Kodak SO-163 film
    EM image scansNumber digital images: 106 / Od range: 1 / Quant bit size: 16 / Sampling size: 12.9 microns / Scanner model: OTHER
    RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
    Radiation wavelengthRelative weight: 1

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    Processing

    Image selectionSoftware name: Situs
    EM single particle entitySymmetry type: ASYMMETRIC
    3D reconstructionMethod: multi-reference projection matching / Software: Spider / Resolution: 36 A / Resolution method: FSC at 0.5 cut-off / Number of particles: 3146 / CTF correction method: each micrograph / Details: The particles were selected interactively.
    Atomic model buildingSoftware name: Situs / Details: the TFIIS chain S was removed before fitting / Ref protocol: rigid body / Ref space: REAL / Target criteria: contour-based Laplacian correlation
    Number of atoms included #LASTProtein: 31127 / Nucleic acid: 0 / Ligand: 10 / Solvent: 0 / Total: 31137

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