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- PDB-7pp4: Cryo-EM structure of Mycobacterium tuberculosis RNA polymerase ho... -

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Basic information

Entry
Database: PDB / ID: 7pp4
TitleCryo-EM structure of Mycobacterium tuberculosis RNA polymerase holoenzyme comprising sigma factor SigB
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor SigB
KeywordsTRANSCRIPTION / DNA-dependent RNA polymerase / alternative sigma
Function / homology
Function and homology information


RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat ...RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / response to hypoxia / protein dimerization activity / response to xenobiotic stimulus / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 ...Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / 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 / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
RNA polymerase sigma factor SigB / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsBrodolin, K.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-16-CE11-0025 France
Citation
Journal: Biorxiv / Year: 2022
Title: Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization
Authors: Morichaud, Z. / Trapani, S. / Vishwakarma, R. / Chaloin, L. / Lionne, C. / Lai-Kee-Him, J. / Bron, P. / Brodolin, K.
#1: Journal: Acta Crystallogr., Sect. D: Biol. Crystallogr. / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography
Authors: Afonine, P.V. / Poon, B.K. / Read, R.J. / Sobolev, O.V. / Terwilliger, T.C. / Urzhumtsev, A. / Adams, P.D.
#2: Journal: Nucleic Acids Res / Year: 2014
Title: Mycobacterium RbpA cooperates with the stress-response σB subunit of RNA polymerase in promoter DNA unwinding.
Authors: Yangbo Hu / Zakia Morichaud / Ayyappasamy Sudalaiyadum Perumal / Françoise Roquet-Baneres / Konstantin Brodolin /
Abstract: RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. ...RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σ(A) subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σ(A) and stress-response σ(B), but not the alternative σ subunits from the groups 3 and 4. In contrast to σ(A), the σ(B) subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response.
History
DepositionSep 13, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release

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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
f: RNA polymerase sigma factor SigB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)403,4709
Polymers403,3156
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area32770 Å2
ΔGint-178 kcal/mol
Surface area136950 Å2

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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: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: rpoA, Rv3457c, MTCY13E12.10c / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGZ1, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129602.344 Da / Num. of mol.: 1 / Mutation: L2E3G4C5 -> V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: rpoB, Rv0667, MTCI376.08c / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147797.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: contains C-terminal 6xHis-tag
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: rpoC, Rv0668, MTCI376.07c / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: rpoZ, Rv1390, MTCY21B4.07 / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY5, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules f

#5: Protein RNA polymerase sigma factor SigB


Mass: 38572.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: contains N-terminal 6xHist-tag
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: sigB, mysB, Rv2710 / Plasmid: pET28a
Details (production host): derivative containing Rv2710 gene
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGI5

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Non-polymers , 2 types, 3 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

ComponentName: RNA polymerase holoenzyme with sigma factor SigB / Type: COMPLEX
Details: RNA polymerase holoenzyme assembled from individually expressed RNA polymerase core and sigma factor SigB
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 DE3 / Plasmid: pET28a
Buffer solutionpH: 7.9
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 49.6 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3064

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19_4092refinement
PHENIX1.19_4092refinement
EM software
IDNameCategory
2Leginonimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115112 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6FBV

6fbv

RefinementHighest resolution: 3.84 Å / Cross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 64.12 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002424219
ELECTRON MICROSCOPYf_angle_d0.482932799
ELECTRON MICROSCOPYf_chiral_restr0.04013723
ELECTRON MICROSCOPYf_plane_restr0.00324332
ELECTRON MICROSCOPYf_dihedral_angle_d9.42759232

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