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- PDB-7qxi: Cryo-EM structure of RNA polymerase-sigma54 holo enzyme with prom... -

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Basic information

Entry
Database: PDB / ID: 7qxi
TitleCryo-EM structure of RNA polymerase-sigma54 holo enzyme with promoter DNA closed complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-Template promoter DNA
  • RNA polymerase sigma-54 factor
  • Template DNA promoter
KeywordsTRANSCRIPTION / Cryo EM / RNA polymerase / transcription close complex / sigma54
Function / homology
Function and homology information


DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / 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 ...DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / 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 / nucleotidyltransferase activity / DNA-directed RNA polymerase complex / 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 / intracellular iron ion homeostasis / transcription cis-regulatory region binding / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytoplasm / cytosol
Similarity search - Function
Sigma-54 factors family signature 1. / 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. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54 ...Sigma-54 factors family signature 1. / 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. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54 / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase Rpb1 funnel domain / Topoisomerase I; Chain A, domain 4 / DNA-directed RNA polymerase, insert domain / RNA Polymerase Alpha Subunit; Chain 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / Beta Complex / 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 / Alpha-Beta Complex / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / 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: 3.4 Å
AuthorsYe, F.Z. / Zhang, X.D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N007816/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/P007503/1 United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: Mechanisms of DNA opening revealed in AAA+ transcription complex structures.
Authors: Fuzhou Ye / Forson Gao / Xiaojiao Liu / Martin Buck / Xiaodong Zhang /
Abstract: Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter ...Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo-electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ inhibition while helping to open up DNA, using σ amino-terminal peptide as a pry bar.
History
DepositionJan 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation.year
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
M: RNA polymerase sigma-54 factor
N: Non-Template promoter DNA
T: Template DNA promoter


Theoretical massNumber of molelcules
Total (without water)484,5638
Polymers484,5638
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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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 / 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) / Strain: K12 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Plasmid: pGEXABC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / 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) / Strain: K12
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Plasmid: pGEXABC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / 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) / Strain: K12 / Gene: rpoC, tabB, b3988, JW3951 / Plasmid: pGEXABC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / 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) / Strain: K12 / Gene: rpoZ, b3649, JW3624 / Plasmid: paCYCDuet-omega / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules M

#5: Protein RNA polymerase sigma-54 factor


Mass: 56165.027 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria)
Gene: rpoN, B4U61_10775, B5L96_24890, BANRA_01956, BANRA_03272, BANRA_04880, BL124_00020515, BN49_0508, BS595_02095, BVX91_19500, C3F39_17700, DBX64_03930, DD583_08830, DDJ63_09965, DRB11_15360, ...Gene: rpoN, B4U61_10775, B5L96_24890, BANRA_01956, BANRA_03272, BANRA_04880, BL124_00020515, BN49_0508, BS595_02095, BVX91_19500, C3F39_17700, DBX64_03930, DD583_08830, DDJ63_09965, DRB11_15360, EHZ17_05145, FXN67_08250, G5637_17550, G7Z27_02650, GJJ08_002620, GNG14_08115, GTH21_20225, NCTC13465_03419, NCTC3279_03959, NCTC5047_03164, SAMEA3499874_03190, SAMEA3499901_03507, SAMEA3512100_03793, SAMEA3538828_04129, SAMEA3649466_03329, SAMEA3649648_01442, SAMEA3649733_03935, SAMEA3649758_00080, SAMEA3720909_03353, SAMEA3727643_03388, SAMEA3727679_00080, SAMEA3729663_03804, SAMEA4364603_00630
Plasmid: pET28b-sigma54 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0N9UTC1

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DNA chain , 2 types, 2 molecules NT

#6: DNA chain Non-Template promoter DNA


Mass: 19439.387 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)
#7: DNA chain Template DNA promoter


Mass: 19404.410 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (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 RNA polymerase-sigma54 holo enzyme with promoter DNA closed complexCOMPLEXall0MULTIPLE SOURCES
2DNA-directed RNA polymerase subunits alpha, beta, beta' and omegaCOMPLEX#1-#41RECOMBINANT
3RNA polymerase sigma-54 factorCOMPLEX#51RECOMBINANT
4Non-Template promoter DNACOMPLEX#61RECOMBINANT
5Template DNA promoterCOMPLEX#71RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Klebsiella pneumoniae (bacteria)573
34Klebsiella pneumoniae (bacteria)573
45Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli BL21(DE3) (bacteria)469008
34Synthetic construct (others)32630
45Synthetic construct (others)32630
Buffer solutionpH: 8
Details: 20 mM Tris-HCl pH 8.0, 150 mM KCl, 10 mM MgCl2,8 mM CHAPSO
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
28 mMCHAPSOC32H58N2O7S1
3150 mMpotassium chlorideKCL1
410 mMMagnesium chlorideMgCL21
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was mxied in vitro in an order to form complex
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1400 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14780
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
4GctfGCTF_v1.18_sm30-75_cu10.1CTF correction
7UCSF Chimerachimera/1.13.1model fitting
8CootCoot/0.8.9.2model fitting
10PHENIXphenix/1.13-2998model refinement
11REFMACccp4/7.0 software packagemodel refinement
12RELIONRelion3.1initial Euler assignment
13RELIONRelion3.1final Euler assignment
14RELIONRelion3.1classification
15RELIONRelion3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2200000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29321 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL
Atomic model buildingPDB-ID: 5NSR

5nsr


Accession code: 5NSR / Source name: PDB / Type: experimental model

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