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- PDB-8qti: Mycobacterium smegnatis RNAP open promoter complex with SigmaA an... -

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Basic information

Entry
Database: PDB / ID: 8qti
TitleMycobacterium smegnatis RNAP open promoter complex with SigmaA and RbpA
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA 50-mer non-template strand
  • DNA 50-mer template strand
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
KeywordsTRANSCRIPTION / HelD / RNA polymerase / transcription initiation / Mycobacteria / rifampicin resistance
Function / homology
Function and homology information


bacterial-type RNA polymerase core enzyme binding / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / DNA-templated transcription ...bacterial-type RNA polymerase core enzyme binding / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 ...RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / 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 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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, 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsKoval, T. / Krasny, L. / Dohnalek, J. / Kouba, T.
Funding support Czech Republic, European Union, 5items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic23-06295S Czech Republic
Ministry of Education (MoE, Czech Republic)LM2023042 Czech Republic
Czech Academy of Sciences86652036 Czech Republic
Grant Agency of the Czech Republic23-06295S Czech Republic
European Union (EU)LX22NPO5103European Union
CitationJournal: To Be Published
Title: Molecular insights into RNA polymerase recycling by mycobacterial HelD
Authors: Koval, T. / Dohnalek, L. / Libor, K. / Kouba, T.
History
DepositionOct 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
F: RNA polymerase sigma factor SigA
O: DNA 50-mer non-template strand
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
J: RNA polymerase-binding protein RbpA
P: DNA 50-mer template strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)458,59612
Polymers458,4419
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 37959.441 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: A0QSL8, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 128680.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: P60281, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 146712.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: A0QS66
#6: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11544.763 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rpoZ, MSMEG_3053, MSMEI_2977 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QWT1, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules FJ

#3: Protein RNA polymerase sigma factor SigA


Mass: 51573.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: sigA, MSMEG_2758 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QW02
#7: Protein RNA polymerase-binding protein RbpA


Mass: 13078.731 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rbpA, MSMEG_3858, MSMEI_3768 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QZ11

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DNA chain , 2 types, 2 molecules OP

#4: DNA chain DNA 50-mer non-template strand


Mass: 15521.958 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycolicibacterium smegmatis MC2 155 (bacteria)
#8: DNA chain DNA 50-mer template strand


Mass: 15409.913 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycolicibacterium smegmatis MC2 155 (bacteria)

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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: Mycobacterium smegnatis RNAP open promoter complex with SigmaA and RbpA
Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELD / Nominal defocus max: 2900 nm / Nominal defocus min: 500 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
11RELION4final Euler assignment
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111613 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00228508
ELECTRON MICROSCOPYf_angle_d0.46438991
ELECTRON MICROSCOPYf_dihedral_angle_d14.7264568
ELECTRON MICROSCOPYf_chiral_restr0.044434
ELECTRON MICROSCOPYf_plane_restr0.0034807

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