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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 / homology
Function and homology information


nitrogen fixation / DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility ...nitrogen fixation / DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation / nucleotidyltransferase activity / transcription elongation factor complex / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Sigma-54 factors family signature 1. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / RNA polymerase sigma factor 54 ...Sigma-54 factors family signature 1. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / RNA polymerase sigma factor 54 / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / 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, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / Beta Complex / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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 / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA polymerase sigma-54 factor / RNA polymerase factor sigma-54 / RNA polymerase sigma-54 factor / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsGlyde, R. / Ye, F.Z. / Zhang, X.D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/N007816 United Kingdom
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.
History
DepositionMay 2, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 4, 2018Provider: repository / Type: Initial release

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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
Polymers484,5109
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42560 Å2
ΔGint-231 kcal/mol
Surface area188200 Å2
MethodPISA

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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 / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
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 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 method: isolated from a genetically manipulated source
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 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 method: isolated from a genetically manipulated source
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 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 method: isolated from a genetically manipulated source
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 method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)
#6: DNA chain NifH promoter non-template DNA


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

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Protein / RNA chain , 2 types, 2 molecules MR

#7: Protein RNA polymerase sigma-54 factor


Mass: 54767.043 Da / Num. of mol.: 1 / Mutation: R336A,R336A,R336A,R336A,R336A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: SM87_03359, CP995_28845 / Plasmid: pET28b-sigma54
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0J4U551, UniProt: A0A2A5PML4, UniProt: P06223*PLUS, DNA-directed RNA polymerase
#8: RNA chain de novo synthesized RNA


Mass: 1296.826 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 / 3D 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 complexCOMPLEXall0MULTIPLE SOURCES
2DNA-directed RNA polymerasePolymeraseCOMPLEX#1-#41RECOMBINANT
3Promoter DNACOMPLEX#5-#61RECOMBINANT
4RNA polymerase sigma-54 factorCOMPLEX#71RECOMBINANT
5de novo DNACOMPLEX#81RECOMBINANT
Molecular weightValue: 0.490 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli K-12 (bacteria)83333
33Klebsiella pneumoniae (bacteria)573
44Klebsiella pneumoniae (bacteria)573
55Escherichia coli K-12 (bacteria)83333
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
33synthetic construct (others)32630
44Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
55Escherichia coli K-12 (bacteria)83333
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris hydrochlorideTris-HCLTris1
2150 mMsodium chlorideNaCLSodium chloride1
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 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89996 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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