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Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

by single particle reconstruction, at 19.8 A resolution

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#2: Superimposing with EM 3D map: EMDB-5127, Image by UCSF CHIMERA

Entry
Summary
Database / IDPORTEIN DATA BANK (PDB) / 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
AuthorsHudson, B.P., Quispe, J., Lara, S., Kim, Y., Berman, H., Arnold, E., Ebright, R.H., Lawson, C.L.
DateDeposition: 2009-08-01, Release: 2009-11-10
PDBj Mine pagesSummary, Structural Details, Experimental Details, Functional Details
Other databasesRCSB-PDB, PDBe, FSSP, SCOP
Structure Visualization
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#1: Depositted structure unit, Image by Jmol

#2: Superimposing with EM 3D map: EMDB-5127, Image by UCSF CHIMERA

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EMDB-5127

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Article
Citation - primary
ArticleProc. Natl. Acad. Sci. U.S.A., Vol. 106, Issue 47, Page 19830-5, Year 2009
TitleThree-dimensional EM structure of an intact activator-dependent transcription initiation complex.
AuthorsBrian P Hudson, Joel Quispe, Samuel Lara-González, Younggyu Kim, Helen M Berman, Eddy Arnold, Richard H Ebright, Catherine L Lawson
Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854, USA.
KeywordsBase Sequence, Cyclic AMP Receptor Protein (chemistry), DNA, Bacterial (chemistry), DNA-Directed RNA Polymerases (chemistry, 2.7.7.6), Escherichia coli Proteins (chemistry), Macromolecular Substances (chemistry), Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Promoter Regions, Genetic, Protein Structure, Tertiary, Protein Subunits (chemistry), Transcription, Genetic
LinksDOI: 10.1073/pnas.0908782106, PubMed: 19903881, PMC: PMC2775702
Components
ID 1 : RNAP subunit alpha, Transcriptase subunit alpha, RNA polymerase subunit alpha
Image
DescriptionDNA-directed RNA polymerase subunit alpha
Typepolypeptide(L)
FragmentRNAP polymerase (sigma70 holoenzyme)
Formula weight36559.020 Da
Number of molecules2
ID1
Ec2.7.7.6
SourceMethod: Isolated from a genetically manipulated source
Gene: K-12, b3295, JW3257, pez, phs, rpoA, RpoB, RpoC, RpoD, RpoZ, sez, ID:83333, Escherichia coli
Host: ID:562, Escherichia coli

, BL21(DE2)
Plasmid name: pEcABC-H6, pRSFduet-sigma, pCDF-omega
LinksUniProt: P0A7Z4, Sequence view
ID 2 : RNAP subunit beta, Transcriptase subunit beta, RNA polymerase subunit beta
Image
DescriptionDNA-directed RNA polymerase subunit beta
Typepolypeptide(L)
FragmentCatabolite activator protein
Formula weight150821.906 Da
Number of molecules1
ID2
Ec2.7.7.6
SourceMethod: Isolated from a genetically manipulated source
Gene: K-12, rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950, ID:83333, Escherichia coli
Host: ID:562, Escherichia coli, BL21(DE3)
LinksUniProt: P0A8V2, Sequence view
ID 3 : RNAP subunit beta', Transcriptase subunit beta', RNA polymerase subunit beta'
Image
DescriptionDNA-directed RNA polymerase subunit beta
Typepolypeptide(L)
FragmentLac(ICAP)UP-UV5-BUBBLE
Formula weight156196.594 Da
Number of molecules1
ID3
Ec2.7.7.6
SourceMethod: Obtained synthetically
LinksUniProt: P0A8T7, Sequence view
ID 4 : RNAP omega subunit, Transcriptase subunit omega, RNA polymerase omega subunit
Image
DescriptionDNA-directed RNA polymerase subunit omega
Typepolypeptide(L)
Formula weight10118.456 Da
Number of molecules1
ID4
Ec2.7.7.6
SourceMethod: Isolated from a genetically manipulated source
Gene: rpoZ, b3649, JW3624, ID:83333, Escherichia coli K-12
Host: ID:562, Escherichia coli
LinksUniProt: P0A800, Sequence view
ID 5 : RNA polymerase sigma factor rpoD
Image
DescriptionRNA polymerase sigma factor rpoD
Typepolypeptide(L)
Formula weight70326.766 Da
Number of molecules1
ID5
SourceMethod: Isolated from a genetically manipulated source
Gene: rpoD, ID:83333, Escherichia coli K-12
Host: ID:562, Escherichia coli
LinksUniProt: P00579, Sequence view
ID 6 : cAMP receptor protein, cAMP regulatory protein
Image
DescriptionCatabolite gene activator
Typepolypeptide(L)
Formula weight23541.408 Da
Number of molecules2
ID6
SourceMethod: Isolated from a genetically manipulated source
Gene: crp, cap, csm, b3357, JW5702, ID:83333, Escherichia coli K-12
Host: ID:562, Escherichia coli
LinksUniProt: P0ACJ8, Sequence view
ID 7 : DNA (98-MER)
Image
DescriptionDNA (98-MER)
Typepolydeoxyribonucleotide
Formula weight30220.646 Da
Number of molecules1
ID7
SourceMethod: Obtained synthetically
LinksSequence view
ID 8 : DNA (98-MER)
Image
DescriptionDNA (98-MER)
Typepolydeoxyribonucleotide
Formula weight30097.557 Da
Number of molecules1
ID8
SourceMethod: Obtained synthetically
LinksSequence view
ID 9 : ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
Image
DescriptionADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
Typenon-polymer
Formula weight329.208 Da
Number of molecules2
ID9
SourceMethod: Obtained synthetically
Sample
Assembly
Aggregation statePARTICLE
CompositionOne molecule of RNAP (containing six subunits) and one CAP homodimer bound to a DNA duplex
DetailsComplex formation was verified by gel shift
Mass-estimation MethodCalculation
Theoretical Mass0.57
NameE. 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
Num components3
Entity assembly
Assembly-ID1
GO-IDGO:00006351
NameRNA polymerase holoenzyme (sigma70)
TypePROTEIN
Entity assembly
Assembly-ID1
Namelac(ICAP)UP-UV5-bubble
TypeNUCLEIC ACID
Entity assembly
Assembly-ID1
GO-IDGO:0006355
InterPro-IDIPR:001808
NameCatabolite Activator Protein
TypePROTEIN
Buffer
Name25 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM cAMP
Experiment
Reconstruction methodSINGLE PARTICLE
Specimen typeNEGATIVE STAINING
Sample preparation
Details25mM HEPES, 100mM KCl, 10mM MgCl2, 1mM DTT, 0.2mM cAMP
pH8.0
Sample concentration6.18 mg/ml
Sample support
Details400-mesh copper 2.0x0.5 hole pattern C-flat grids (Protochips, Inc.) with a thin layer of continuous carbon floated on top
Vitrification
Cryogen nameNONE
Experiment
MethodELECTRON MICROSCOPY
Electron Microscopy
Imaging
MicroscopeModel: FEI TECNAI F20
Date4-NOV-2008
Details15 um pixel size on detector
Electron gun
Electron sourceFIELD EMISSION GUN
Accelerating voltage120 kV
Electron dose16 e/A**2
Illumination modeFLOOD BEAM
Lens
ModeBRIGHT FIELD
MagnificationNominal: 50000 X
CsNominal: 2.0 mm
Detector distance0.0
Nominal defocusMax: 1500 nm, Min: 500 nm
Specimen holder
Specimen holderModel: SIDE ENTRY, EUCENTRIC, Type: standard side-entry room-temperature stage
Tilt angleMin: 0.0 degrees, Max: 0.0 degrees
Temperature293 Kelvin
Detector
TypeTeitz F415 4k x 4k pixel CCD camera
Processing
2D projection selection
Software nameChimera, Yup.scx
Single particle entity
Symmetry typeASYMMETRIC
3D reconstruction
CTF correction methodACE
DetailsEMAN interleaved with SPIDER correspondence analysis ( Details about the particle: 32816 particles were automatically selected by the Appion DoGpicker initially )
Methodprojection matching
Num class averages280
Number of particles14097
Resolution19.8 A
Resolution methodFSC at 0.5 cut-off
SoftwareEMAN, SPIDER
3D fitting
DetailsA 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.
Refinement Protocolrigid body, Yup.scx simulated annealing
Refinement SpaceREAL
Software nameChimera, Yup.scx
Target criteriamap-derived potential energy
3D fitting
DetailsA 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.
Refinement Protocolrigid body fit followed by Yup.scx simulated annealing
Refinement SpaceREAL
Software nameChimera, Yup.scx
Target criteriamap-derived potential energy
3D fitting
DetailsA 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.
Refinement Protocolmanual fit followed by Yup.scx simulated annealing
Refinement SpaceREAL
Software nameChimera, Yup.scx
Target criteriamap-derived potential energy
3D fitting
DetailsA 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.
Refinement Protocolrigid body fit followed by Yup.scx simulated annealing
Refinement SpaceREAL
Software nameChimera, Yup.scx
Target criteriamap-derived potential energy
3D fitting
DetailsA 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.
Refinement Protocolrigid body fit followed by Yup.scx simulated annealing
Refinement SpaceREAL
Software nameChimera, Modeller, Yup.scx
Target criteriamap-derived potential energy
3D fitting list
3D Fitting ID1
3D fitting list
3D Fitting ID2
3D fitting list
3D Fitting ID3
Pdb chain idA
3D fitting list
3D Fitting ID4
Pdb chain idA
3D fitting list
3D Fitting ID5
Refine hist
Cycle idLAST
Refine idELECTRON MICROSCOPY
Total atoms35254
Ligand atoms44
Nucleic acid atoms4002
Protein atoms31208
Download
PDB format
Allpdb3iyd.ent.gz
pdb3iyd.ent (uncompressed file)
Header onlypdb3iyd.ent.gz
mmCIF format
mmCIF3iyd.cif.gz
XML format
All3iyd.xml.gz
No-atom3iyd-noatom.xml.gz
Ext-atom3iyd-extatom.xml.gz
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