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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 / エシェリキア・コリ, 大腸菌 /
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
Remark 650HELIX DETERMINATION METHOD: AUTHOR DETERMINED
Remark 700SHEET DETERMINATION METHOD: AUTHOR DETERMINED

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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


Theoretical massNumber of molelcules
Total (without water)568,64112
Polyers567,98310
Non-polymers6582
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Polypeptide(L) , 6 types, 8 molecules ABCDEFGH

#1: Polypeptide(L)DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / Transcriptase subunit alpha / RNA polymerase subunit alpha


Mass: 36559.020 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli / References: UniProt: P0A7Z4, EC: 2.7.7.6

Cellular component

Molecular function

Biological process

#2: Polypeptide(L)DNA-directed RNA polymerase subunit beta / RNAP subunit beta / Transcriptase subunit beta / RNA polymerase subunit beta


Mass: 150821.906 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / References: UniProt: P0A8V2, EC: 2.7.7.6

Cellular component

Molecular function

Biological process

#3: Polypeptide(L)DNA-directed RNA polymerase subunit beta / RNAP subunit beta' / Transcriptase subunit beta' / RNA polymerase subunit beta'


Mass: 156196.594 Da / Num. of mol.: 1 / References: UniProt: P0A8T7, EC: 2.7.7.6

Cellular component

Molecular function

Biological process

#4: Polypeptide(L)DNA-directed RNA polymerase subunit omega / RNAP omega subunit / Transcriptase subunit omega / RNA polymerase omega subunit


Mass: 10118.456 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli K-12 / References: UniProt: P0A800, EC: 2.7.7.6

Cellular component

Molecular function

Biological process

#5: Polypeptide(L)RNA polymerase sigma factor rpoD / RNAP polymerase (sigma70 holoenzyme)


Mass: 70326.766 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli K-12 / References: UniProt: P00579

Cellular component

Molecular function

Biological process

  • response to heat (GO: 0009408)
  • transcription initiation from bacterial-type RNA polymerase promoter (GO: 0001123)
#6: Polypeptide(L)Catabolite gene activator / cAMP receptor protein / cAMP regulatory protein / Catabolite activator protein


Mass: 23541.408 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli K-12 / References: UniProt: P0ACJ8

Cellular component

Molecular function

Biological process

  • carbon catabolite repression of transcription (GO: 0045013)
  • negative regulation of transcription, DNA-templated (GO: 0045892)
  • positive regulation of transcription, DNA-templated (GO: 0045893)
  • transcription, DNA-templated (GO: 0006351)

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DNA chain , 2 types, 2 molecules IJ

#7: DNA chainDNA (98-MER) / Lac(ICAP)UP-UV5-BUBBLE


Mass: 30220.646 Da / Num. of mol.: 1
#8: DNA chainDNA (98-MER)


Mass: 30097.557 Da / Num. of mol.: 1

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Non-polymers , 1 types, 2 molecules

#9: ChemicalChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.208 Da / Num. of mol.: 2 / Formula: C10H12N5O6P

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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

IDNameTypeGo id
3RNA polymerase holoenzyme (sigma70)PROTEINGO:00006351
2lac(ICAP)UP-UV5-bubbleNUCLEIC ACID
1Catabolite Activator ProteinPROTEINGO: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
1LB211
1BDF22
2AUKA33
1SIGA44
3DXJ55
Number of atoms included #LASTProtein: 31208 / Nucleic acid: 4002 / Ligand: 44 / Solvent: 0 / Total: 35254

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