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    - PDB-3iyd: Three-dimensional EM structure of an intact activator-dependent t... -

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    Basic information

    Entry
    Database: PDB / ID: 3iyd
    TitleThree-dimensional EM structure of an intact activator-dependent transcription initiation complex
    DescriptorRNA polymerase sigma factor rpoD
    Catabolite gene activator/DNA complex
    (DNA-directed RNA polymerase subunit ...) x 3
    KeywordsTRANSCRIPTION/DNA / transcription / initiation / Class I / activator / RNA polymerase / holoenzyme / sigma70 / open complex / CAP / CRP / cAMP-dependent / DNA / prokaryotic / DNA-directed RNA polymerase / Nucleotidyltransferase / Transferase / DNA-binding / Sigma factor / Transcription regulation / cAMP / cAMP-binding / Nucleotide-binding / TRANSCRIPTION-DNA COMPLEX
    Specimen sourceEscherichia coli / bacteria /
    Escherichia coli k-12 / bacteria / image: Escherichia coli
    MethodElectron microscopy (19.8 A resolution / Single particle / Negative staining)
    AuthorsHudson, B.P. / Quispe, J. / Lara, S. / Kim, Y. / Berman, H. / Arnold, E. / Ebright, R.H. / Lawson, C.L.
    CitationProc. Natl. Acad. Sci. U.S.A., 2009, 106, 19830-19835

    Proc. Natl. Acad. Sci. U.S.A., 2009, 106, 19830-19835 StrPapers
    Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.
    Brian P Hudson / Joel Quispe / Samuel Lara-González / Younggyu Kim / Helen M Berman / Eddy Arnold / Richard H Ebright / Catherine L Lawson

    DateDeposition: Aug 1, 2009 / Release: Nov 10, 2009 / Last modification: Feb 10, 2016

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    Assembly

    Deposited unit
    A: DNA-directed RNA polymerase subunit alpha
    B: DNA-directed RNA polymerase subunit alpha
    C: DNA-directed RNA polymerase subunit beta
    D: DNA-directed RNA polymerase subunit beta
    E: DNA-directed RNA polymerase subunit omega
    F: RNA polymerase sigma factor rpoD
    G: Catabolite gene activator
    H: Catabolite gene activator
    I: DNA (98-MER)
    J: DNA (98-MER)
    hetero molecules

    569 kDa, 12 molecules
    Theoretical massNumber of molelcules
    Total
    (without water)
    568,64112
    Polyers567,98310
    Non-polymers6582
    Water0

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

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    Components

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    DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

    #1polypeptide(L) / DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha, Transcriptase subunit alpha, RNA polymerase subunit alpha / Source: Escherichia coli (gene. exp.) / References: UniProt: P0A7Z4, EC: 2.7.7.6
    #2polypeptide(L) / DNA-directed RNA polymerase subunit beta / RNAP subunit beta, Transcriptase subunit beta, RNA polymerase subunit beta / Source: Escherichia coli (gene. exp.) / References: UniProt: P0A8V2, EC: 2.7.7.6
    #3polypeptide(L) / DNA-directed RNA polymerase subunit beta / RNAP subunit beta', Transcriptase subunit beta', RNA polymerase subunit beta' / References: UniProt: P0A8T7, EC: 2.7.7.6
    #4polypeptide(L) / DNA-directed RNA polymerase subunit omega / RNAP omega subunit, Transcriptase subunit omega, RNA polymerase omega subunit / Source: Escherichia coli K-12 (gene. exp.) / References: UniProt: P0A800, EC: 2.7.7.6

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    Polypeptide(L) , 2 types, 3 molecules FGH

    #5polypeptide(L) / RNA polymerase sigma factor rpoD / RNAP polymerase (sigma70 holoenzyme) / Source: Escherichia coli K-12 (gene. exp.) / References: UniProt: P00579
    #6polypeptide(L) / Catabolite gene activator / cAMP receptor protein, cAMP regulatory protein, Catabolite activator protein / Source: Escherichia coli K-12 (gene. exp.) / References: UniProt: P0ACJ8

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    DNA chain , 2 types, 2 molecules IJ

    #7DNA chain / DNA (98-MER) / Lac(ICAP)UP-UV5-BUBBLE
    #8DNA chain / DNA (98-MER)

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    Non-polymers , 1 types, 2 molecules

    #9ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

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    Experimental details

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    Experiment

    ExperimentMethod: ELECTRON MICROSCOPY
    EM experimentReconstruction method: SINGLE PARTICLE / Specimen type: NEGATIVE STAINING

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    Sample preparation

    Assembly of specimenName: E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolite activator protein (CAP) bound to 98-mer DNA containing the lac promoter and engineered open transcription bubble
    Aggregation state: PARTICLE / Details: Complex formation was verified by gel shift
    Composition: One molecule of RNAP (containing six subunits) and one CAP homodimer bound to a DNA duplex
    Mol wt method: Calculation / Mol wt theo: 0.57 MDa / Number of components: 3
    Component

    Assembly id: 1 / Ipr id: IPR001808

    NameTypeIDGo id
    RNA polymerase holoenzyme (sigma70)PROTEIN3GO:00006351
    lac(ICAP)UP-UV5-bubbleNUCLEIC ACID2
    Catabolite Activator ProteinPROTEIN1GO:0006355
    Buffer solutionName: 25 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM cAMP
    Sample preparationDetails: 25mM HEPES, 100mM KCl, 10mM MgCl2, 1mM DTT, 0.2mM cAMP
    pH: 8 / Sample conc.: 6.18 mg/ml
    Specimen supportDetails: 400-mesh copper 2.0x0.5 hole pattern C-flat grids (Protochips, Inc.) with a thin layer of continuous carbon floated on top
    VitrificationCryogen name: NONE

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    Electron microscopy imaging

    EM imagingCamera length: 0 mm
    MicroscopyMicroscope model: FEI TECNAI F20 / Date: Nov 4, 2008 / Details: 15 um pixel size on detector
    Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 16 e/A2 / Illumination mode: FLOOD BEAM
    Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2 mm
    Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC
    Specimen holder type: standard side-entry room-temperature stage
    Temperature: 293 K / Tilt angle max: 0 deg. / Tilt angle min: 0 deg.
    CameraType: Teitz F415 4k x 4k pixel CCD camera
    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: Chimera, Yup.scx
    EM single particle entitySymmetry type: ASYMMETRIC
    3D reconstructionMethod: projection matching / Software: EMAN, SPIDER / Resolution: 19.8 A / Resolution method: FSC at 0.5 cut-off / Number of particles: 14097 / CTF correction method: ACE
    Details: EMAN interleaved with SPIDER correspondence analysis ( Details about the particle: 32816 particles were automatically selected by the Appion DoGpicker initially )
    Number of class averages: 280
    Atomic model building

    Details: A complete ternary complex model was generated using multiple PDB entries, with a homology modelling step for RNAP. The model was regularized with PHENIX and the refined against the EM map with Yup.scx using default parameters. / Ref space: REAL / Target criteria: map-derived potential energy

    Software nameIDRef protocol
    Chimera, Yup.scx1rigid body, Yup.scx simulated annealing
    Chimera, Yup.scx2rigid body fit followed by Yup.scx simulated annealing
    Chimera, Yup.scx3manual fit followed by Yup.scx simulated annealing
    Chimera, Yup.scx4rigid body fit followed by Yup.scx simulated annealing
    Chimera, Modeller, Yup.scx5rigid body fit followed by Yup.scx simulated annealing
    Atomic model building
    PDB-IDPdb chain id 3D fitting idID
    1LB2 11
    1BDF 22
    2AUK A33
    1SIG A44
    3DXJ 55
    Number of atoms included #LASTProtein: 31208 / Nucleic acid: 4002 / Ligand: 44 / Solvent: 0 / Total: 35254

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