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- PDB-5vt0: Escherichia coli 6S RNA derivative in complex with Escherichia co... -

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Basic information

Entry
Database: PDB / ID: 5vt0
TitleEscherichia coli 6S RNA derivative in complex with Escherichia coli RNA polymerase sigma70-holoenzyme
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • Escherichia coli 6S RNA derivative (112-MER)
  • RNA polymerase sigma factor RpoD
Keywordstranscription/RNA / RNAP / 6S RNA / ncRNA / transcription regulation / transcription-RNA complex
Function / homologyDNA-directed RNA polymerase, subunit beta-prime / RNA polymerase sigma factor, region 2 / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 3/4-like / RNA polymerase, RBP11-like subunit ...DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase sigma factor, region 2 / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 3/4-like / RNA polymerase, RBP11-like subunit / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / RNA polymerase, alpha subunit, C-terminal / Winged helix-like DNA-binding domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase beta subunit / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb2, domain 7 / Sigma-70, non-essential region / Sigma-70, region 4 / Sigma-70 region 2 / Sigma-70 region 3 / Sigma-70 factor, region 1.1 / RNA polymerase Rpb3/Rpb11 dimerisation domain / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb6 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb2, domain 6 / Sigma-70 factor, region 1.2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase sigma-70 region 1.2 / RNA polymerase Rpb2, domain 3 / RNA polymerase, N-terminal / RNA polymerases beta chain signature. / Sigma-70 factors family signature 2. / Sigma-70 factors family signature 1. / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase, alpha subunit / RNA polymerase sigma-70 / RNA polymerase, beta subunit, protrusion / RNA polymerase, subunit omega/K/RPB6 / DNA-directed RNA polymerase, omega subunit / RNA polymerase Rpb1, domain 3 / RNA polymerase sigma factor 70, region 1.1 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma factor 70, non-essential domain / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase Rpb1, domain 1 / RNA polymerase sigma-70 region 3 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb2, domain 2 / RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription, elongation / sigma factor activity / ribonucleoside binding / DNA-templated transcription, initiation / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein dimerization activity / DNA-binding transcription factor activity / transcription, DNA-templated / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / 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
Function and homology information
Specimen sourceEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.78 Å resolution
AuthorsChen, J. / Darst, S.A.
CitationJournal: Mol. Cell / Year: 2017
Title: 6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase.
Authors: James Chen / Karen M Wassarman / Shili Feng / Katherine Leon / Andrey Feklistov / Jared T Winkelman / Zongli Li / Thomas Walz / Elizabeth A Campbell / Seth A Darst
Abstract: Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. ...Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ-holoenzyme (Eσ) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 15, 2017 / Release: Nov 1, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 1, 2017Structure modelrepositoryInitial release
1.1Dec 6, 2017Structure modelAuthor supporting evidence / Othercell / pdbx_audit_support_cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
L: RNA polymerase sigma factor RpoD
R: Escherichia coli 6S RNA derivative (112-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)476,02710
Polyers475,8727
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)47140
ΔGint (kcal/M)-246
Surface area (Å2)174630
MethodPISA

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Components

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

#1: Protein/peptide DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 26330.996 Da / Num. of mol.: 2
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein/peptide DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein/peptide DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein/peptide DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein/peptide / RNA chain , 2 types, 2 molecules LR

#5: Protein/peptide RNA polymerase sigma factor RpoD / Sigma-70


Mass: 60464.371 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P00579
#6: RNA chain Escherichia coli 6S RNA derivative (112-MER)


Mass: 46308.500 Da / Num. of mol.: 1 / Details: prepared by in vitro transcription / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 2 types, 3 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Escherichia coli 6S RNA derivative in complex with Escherichia coli RNA polymerase sigma70-holoenzyme
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / Source: RECOMBINANT
Molecular weightValue: 0.475 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.8 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryDetails
7Coot1.10.10-2155model fitting0.8
9PHENIX1.10.1-2155model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 362926 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL / Target criteria: Correlation coefficient
Least-squares processHighest resolution: 3.78 Å
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00731911
ELECTRON MICROSCOPYf_angle_d1.02743624
ELECTRON MICROSCOPYf_dihedral_angle_d6.41419425
ELECTRON MICROSCOPYf_chiral_restr0.0615063
ELECTRON MICROSCOPYf_plane_restr0.0075286

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