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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
Components
  • (DNA (98-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Catabolite gene activator
  • RNA polymerase sigma factor rpoD
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
Function / homology
Function and homology information


carbon catabolite repression of transcription / sigma factor antagonist complex / DNA binding, bending / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / minor groove of adenine-thymine-rich DNA binding / sigma factor activity / bacterial-type flagellum assembly ...carbon catabolite repression of transcription / sigma factor antagonist complex / DNA binding, bending / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / minor groove of adenine-thymine-rich DNA binding / sigma factor activity / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / cAMP binding / regulation of DNA-templated transcription elongation / transcription elongation factor complex / transcription antitermination / cell motility / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / response to heat / protein-containing complex assembly / sequence-specific DNA binding / intracellular iron ion homeostasis / protein dimerization activity / DNA-binding transcription factor activity / response to antibiotic / negative regulation of DNA-templated transcription / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / identical protein binding / membrane / cytoplasm / cytosol
Similarity search - Function
helix_turn_helix, cAMP Regulatory protein / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / : / Crp-like helix-turn-helix domain / Crp-type HTH domain profile. / Crp-type HTH domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 ...helix_turn_helix, cAMP Regulatory protein / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / : / Crp-like helix-turn-helix domain / Crp-type HTH domain profile. / Crp-type HTH domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / RmlC-like jelly roll fold / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / DNA / DNA (> 10) / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-binding transcriptional dual regulator CRP
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 19.8 Å
AuthorsHudson, B.P. / Quispe, J. / Lara, S. / Kim, Y. / Berman, H. / Arnold, E. / Ebright, R.H. / Lawson, C.L.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2009
Title: Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.
Authors: Brian P Hudson / Joel Quispe / Samuel Lara-González / Younggyu Kim / Helen M Berman / Eddy Arnold / Richard H Ebright / Catherine L Lawson /
Abstract: We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA ...We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.
History
DepositionAug 1, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 10, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Source and taxonomy / Version format compliance
Revision 1.2Feb 10, 2016Group: Structure summary
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 650HELIX DETERMINATION METHOD: AUTHOR DETERMINED
Remark 700SHEET DETERMINATION METHOD: AUTHOR DETERMINED

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-5127
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
Polymers567,97910
Non-polymers6582
Water00
1


  • Idetical with deposited unit
  • defined by author
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: Protein 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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K-12
Gene: b3295, JW3257, pez, phs, rpoA, RpoB, RpoC, RpoD, RpoZ, sez
Plasmid: pEcABC-H6, pRSFduet-sigma, pCDF-omega / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE2) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein 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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K-12
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta' / Transcriptase subunit beta' / RNA polymerase subunit beta'


Mass: 156195.625 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Purchased from IDT and annealed / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein 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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein , 2 types, 3 molecules FGH

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


Mass: 70326.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoD / Production host: Escherichia coli (E. coli) / References: UniProt: P00579
#6: Protein Catabolite gene activator / cAMP receptor protein / cAMP regulatory protein / Catabolite activator protein


Mass: 23541.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: crp, cap, csm, b3357, JW5702 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ACJ8

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DNA chain , 2 types, 2 molecules IJ

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


Mass: 30220.389 Da / Num. of mol.: 1 / Source method: obtained synthetically
#8: DNA chain DNA (98-MER)


Mass: 30097.295 Da / Num. of mol.: 1 / Source method: obtained synthetically

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Non-polymers , 1 types, 2 molecules

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


Mass: 329.206 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H12N5O6P

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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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 MDa / Experimental value: NO
Buffer solutionName: 25 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM cAMP
pH: 8
Details: 25 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM cAMP
SpecimenConc.: 6.18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
Details: 25mM HEPES, 100mM KCl, 10mM MgCl2, 1mM DTT, 0.2mM cAMP
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
MicroscopyModel: 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 X / 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 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 16 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (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
4EMAN3D reconstruction
5SPIDER3D reconstruction
CTF correctionDetails: ACE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: projection matching / Resolution: 19.8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 14097
Details: EMAN interleaved with SPIDER correspondence analysis ( Details about the particle: 32816 particles were automatically selected by the Appion DoGpicker initially )
Num. of class averages: 280 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: map-derived potential energy
Details: 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 ...Details: 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.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
11LB2

1lb2

1LB21
21BDF

1bdf

1BDF2
32AUK

2auk

A2AUK3
41SIG

1sig

A1SIG4
53DXJ

3dxj

3DXJ5
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms31208 4002 44 0 35254

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