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- PDB-6c6t: CryoEM structure of E.coli RNA polymerase elongation complex boun... -

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Basic information

Entry
Database: PDB / ID: 6c6t
TitleCryoEM structure of E.coli RNA polymerase elongation complex bound with RfaH
Components
  • (DNA (29-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA (5'-R(*GP*CP*AP*UP*UP*CP*AP*AP*AP*GP*CP*CP*GP*AP*GP*AP*GP*GP*UP*A)-3')
  • Transcription antitermination protein RfaH
Keywordstranscription/dna/rna / RNAP / elongation complex / anti-pausing / TRANSCRIPTION / transcription-dna-rna complex
Function / homologyDNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 3 / Transcription antitermination protein RfaH / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega ...DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 3 / Transcription antitermination protein RfaH / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb2, domain 7 / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb6 / RNA polymerase Rpb3/Rpb11 dimerisation domain / Transcription termination factor nusG / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 2 / RNA polymerases beta chain signature. / 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 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / NusG, N-terminal / RNA polymerase, N-terminal / RNA polymerase, subunit omega/K/RPB6 / Bacterial RNA polymerase, alpha chain C terminal domain / transcription elongation from bacterial-type RNA polymerase promoter / transcription antitermination factor activity, DNA binding / bacterial-type RNA polymerase core enzyme binding / translation activator activity / RNA polymerase complex / transcription antitermination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / positive regulation of translation / protein-containing complex assembly / protein dimerization activity / transcription, DNA-templated / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Transcription antitermination protein RfaH
Function and homology information
Specimen sourceEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.5 Å resolution
AuthorsKang, J.Y. / Artsimovitch, I. / Landick, R. / Darst, S.A.
CitationJournal: Cell / Year: 2018
Title: Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators.
Authors: Jin Young Kang / Rachel Anne Mooney / Yuri Nedialkov / Jason Saba / Tatiana V Mishanina / Irina Artsimovitch / Robert Landick / Seth A Darst
Abstract: NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most ...NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 19, 2018 / Release: Jul 25, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 25, 2018Structure modelrepositoryInitial release
1.1Oct 24, 2018Structure modelData collection / Database referencescitation / citation_author_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Assembly

Deposited unit
A: DNA (29-MER)
B: DNA (29-MER)
R: RNA (5'-R(*GP*CP*AP*UP*UP*CP*AP*AP*AP*GP*CP*CP*GP*AP*GP*AP*GP*GP*UP*A)-3')
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
D: Transcription antitermination protein RfaH
hetero molecules


Theoretical massNumber of molelcules
Total (without water)412,18512
Polyers412,0309
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain DNA (29-MER)


Mass: 9001.772 Da / Num. of mol.: 1 / Source: (synth.) Escherichia coli (E. coli)
#2: DNA chain DNA (29-MER)


Mass: 8838.659 Da / Num. of mol.: 1 / Source: (synth.) Escherichia coli (E. coli)

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

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


Mass: 26459.125 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 (E. coli) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: 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 (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#6: 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 (E. coli) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#7: 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 (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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

#3: RNA chain RNA (5'-R(*GP*CP*AP*UP*UP*CP*AP*AP*AP*GP*CP*CP*GP*AP*GP*AP*GP*GP*UP*A)-3')


Mass: 6469.944 Da / Num. of mol.: 1 / Source: (synth.) Escherichia coli (E. coli)
#8: Protein/peptide Transcription antitermination protein RfaH


Mass: 18364.217 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rfaH, hlyT, sfrB, b3842, JW3818 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFW0

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium
#10: 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: E.coli RfaH-elongation complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: RECOMBINANT
Molecular weightValue: 0.412 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
120 mMTrisTris1
2120 mMpotassium acetateKCH3COO1
35 mMmagnesium chlorideMgCl21
45 mMDithiothreitolC4H10O2S21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 kelvins

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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 / Calibrated magnification: 38462 / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 71 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 3495
Image scansMovie frames/image: 50 / Used frames/image: 3-50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION2.0initial Euler assignment
10RELION2.0final Euler assignment
11RELION2.0classification
12RELION2.03D reconstruction
13PHENIX1.12model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 447100
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 174600 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01027461
ELECTRON MICROSCOPYf_angle_d1.10937393
ELECTRON MICROSCOPYf_dihedral_angle_d8.85416643
ELECTRON MICROSCOPYf_chiral_restr0.0664278
ELECTRON MICROSCOPYf_plane_restr0.0084653

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