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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 Å resolution / Particle / Single particle)
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 Yorodumi Papers
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

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 1, 2009 / Release: Nov 10, 2009
RevisionDateData content typeGroupProviderType
1.0Nov 10, 2009Structure modelrepositoryInitial release
1.1Jul 13, 2011Structure modelSource and taxonomy / Version format compliance
1.2Feb 10, 2016Structure modelStructure summary
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)568,63712
Polyers567,97910
Non-polymers6582
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

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


Mass: 36558.680 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: 150820.875 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: 156195.625 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: 10118.352 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

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Polypeptide(L) , 2 types, 3 molecules FGH

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


Mass: 70326.234 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli K-12 / References: UniProt: P00579

Cellular component

Molecular function

Biological process

#6: Polypeptide(L)Catabolite gene activator / cAMP receptor protein / cAMP regulatory protein / Catabolite activator protein


Mass: 23541.242 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: 30220.389 Da / Num. of mol.: 1
#8: DNA chainDNA (98-MER)


Mass: 30097.295 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: 329.206 Da / Num. of mol.: 2 / Formula: C10H12N5O6P

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

Component
IDNameTypeDetailsParent ID
1E. 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 bubbleCOMPLEXOne molecule of RNAP (containing six subunits) and one CAP homodimer bound to a DNA duplex. Complex formation was verified by gel shift0
2Catabolite Activator Protein1
3lac(ICAP)UP-UV5-bubble1
4RNA polymerase holoenzyme (sigma70)1
Molecular weightValue: 0.57 deg. / Units: MEGADALTONS / Experimental value: NO
Buffer solutionName: 25 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM cAMP
Details: 25 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM cAMP
pH: 8
SpecimenConc.: 6.18 mg/ml
Details: 25mM HEPES, 100mM KCl, 10mM MgCl2, 1mM DTT, 0.2mM cAMP
Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: Uranyl Formate
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

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
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 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC
Specimen holder type: standard side-entry room-temperature stage
Temperature: 293 kelvins / Tilt angle max: 0 deg. / Tilt angle min: 0 deg.
Image recordingElectron dose: 16 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1ModellerMODEL FITTING
2UCSF ChimeraMODEL FITTING
3Yup.scxMODEL FITTING
4EMANRECONSTRUCTION
5SPIDERRECONSTRUCTION
CTF correctionDetails: ACE
SymmetryPoint symmetry: C1
3D reconstructionMethod: projection matching / Resolution: 19.8 Å / Resolution method: FSC 0.5 CUT-OFF / Number of particles: 14097
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 / Symmetry type: POINT
Atomic model buildingDetails: REFINEMENT PROTOCOL--rigid body, Yup.scx simulated annealing 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 protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: map-derived potential energy
Atomic model building
IDPDB-IDPdb chain ID 3D fitting ID
11LB21
21BDF1
32AUKA1
41SIGA1
53DXJ1
Number of atoms included #LASTProtein: 31208 / Nucleic acid: 4002 / Ligand: 44 / Solvent: 0 / Total: 35254

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