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- PDB-6gfw: Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme ... -

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Basic information

Entry
Database: PDB / ID: 6gfw
TitleCryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme initial transcribing complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • NifH promoter non-template DNA
  • RNA polymerase sigma-54 factor
  • de novo synthesized RNA
  • nifH promoter template DNA
KeywordsTRANSCRIPTION / transcription initiation / DNA opening / transcription bubble / de novo RNA synthesis
Function / homologyRNA polymerase subunit, RPB6/omega / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain ...RNA polymerase subunit, RPB6/omega / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RPB6/omega subunit-like superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 3 / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / 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 / Bacterial RNA polymerase, alpha chain C terminal domain / Sigma-54, DNA binding domain / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb1, domain 3 / RNA polymerase, subunit omega/K/RPB6 / RNA polymerases beta chain signature. / Sigma-54 factors family signature 2. / Sigma-54 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 Rpb2, domain 2 / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega subunit / RNA polymerase sigma factor 54 / RNA polymerase, N-terminal / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma factor 54, DNA-binding / 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 Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / Sigma-54 factor, core binding domain / RNA polymerase complex / DNA-directed RNA polymerase complex / bacterial-type RNA polymerase transcriptional activator activity, sequence-specific DNA binding / DNA-templated transcription, elongation / sigma factor activity / ribonucleoside binding / DNA-templated transcription, initiation / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / transcription, DNA-templated / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / RNA polymerase sigma-54 factor / RNA polymerase factor sigma-54 / 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 K-12 (bacteria)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.7 Å resolution
AuthorsGlyde, R. / Ye, F.Z. / Zhang, X.D.
CitationJournal: Mol. Cell / Year: 2018
Title: Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation.
Authors: Robert Glyde / Fuzhou Ye / Milija Jovanovic / Ioly Kotta-Loizou / Martin Buck / Xiaodong Zhang
Abstract: Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a ...Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ (σ), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by σ.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 2, 2018 / Release: Jul 4, 2018

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Structure visualization

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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: nifH promoter template DNA
G: NifH promoter non-template DNA
M: RNA polymerase sigma-54 factor
R: de novo synthesized RNA


Theoretical massNumber of molelcules
Total (without water)484,5109
Polyers484,5109
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)42560
ΔGint (kcal/M)-231
Surface area (Å2)188200
MethodPISA

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Components

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

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


Mass: 36558.680 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 K-12 (bacteria)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 K-12 (bacteria) / Gene: rpoC, tabB, b3988, JW3951
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 K-12 (bacteria) / Gene: rpoZ, b3649, JW3624
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules FG

#5: DNA chain nifH promoter template DNA


Mass: 19404.410 Da / Num. of mol.: 1 / Source: (synth.) Klebsiella pneumoniae (bacteria)
#6: DNA chain NifH promoter non-template DNA


Mass: 19487.436 Da / Num. of mol.: 1 / Source: (synth.) Klebsiella pneumoniae (bacteria)

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

#7: Protein/peptide RNA polymerase sigma-54 factor


Mass: 54767.043 Da / Num. of mol.: 1 / Mutation: R336A,R336A,R336A,R336A,R336A / Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: SM87_03359, CP995_28845 / Plasmid name: pET28b-sigma54
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0J4U551, UniProt: A0A2A5PML4, DNA-directed RNA polymerase
#8: RNA chain de novo synthesized RNA


Mass: 1296.826 Da / Num. of mol.: 1 / Details: UGGG / Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Production host: Escherichia coli K-12 (bacteria)

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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

Component
IDNameTypeEntity IDParent IDSource
1Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme initial transcribing complexCOMPLEX1, 2, 3, 4, 5, 6, 7, 80MULTIPLE SOURCES
2DNA-directed RNA polymeraseCOMPLEX1,2,3,41RECOMBINANT
3Promoter DNACOMPLEX5,61RECOMBINANT
4RNA polymerase sigma-54 factorCOMPLEX71RECOMBINANT
5de novo DNACOMPLEX81RECOMBINANT
Molecular weightValue: 0.490 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
2283333Escherichia coli K-12 (bacteria)
33573Klebsiella pneumoniae (bacteria)
44573Klebsiella pneumoniae (bacteria)
5583333Escherichia coli K-12 (bacteria)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
22866768Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
3332630synthetic construct (others)
44866768Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
5583333Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
110 mMTris hydrochlorideTris-HCL1
2150 mMsodium chlorideNaCL1
310 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF Chimera1.9model fitting
8Coot8.03model fitting
10PHENIX1.10.1model refinement
11CCP4 package7model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 89996 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL

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