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- PDB-6vvs: Crystal structure of a Mycobacterium smegmatis RNA polymerase tra... -

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Basic information

Entry
Database: PDB / ID: 6vvs
TitleCrystal structure of a Mycobacterium smegmatis RNA polymerase transcription initiation complex with antibiotic Sorangicin
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (26-MER)
  • DNA (31-MER)
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
  • unknown
KeywordsTRANSCRIPTION / TRANSFERASE/DNA/ANTIBIOTIC / DNA binding / antibiotic / TRANSFERASE-DNA-ANTIBIOTIC complex
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
SORANGICIN A / 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 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.112 Å
AuthorsLilic, M. / Braffman, N. / Darst, S.A. / Campbell, E.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: The antibiotic sorangicin A inhibits promoter DNA unwinding in a rifampicin-resistant RNA polymerase.
Authors: Mirjana Lilic / James Chen / Hande Boyaci / Nathaniel Braffman / Elizabeth A Hubin / Jennifer Herrmann / Rolf Müller / Rachel Mooney / Robert Landick / Seth A Darst / Elizabeth A Campbell /
Abstract: Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen (). The emergence of Rif-resistant (Rif) presents a need for new ...Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen (). The emergence of Rif-resistant (Rif) presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of Rif RNAPs, including the most prevalent clinical Rif RNAP substitution found in infected patients (S456>L of the β subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the Rif S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.
History
DepositionFeb 18, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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 SigA
G: unknown
J: RNA polymerase-binding protein RbpA
O: DNA (31-MER)
P: DNA (26-MER)
T: DNA-directed RNA polymerase subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)487,14635
Polymers484,42911
Non-polymers2,71724
Water77543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area55740 Å2
ΔGint-420 kcal/mol
Surface area147710 Å2
Unit cell
Length a, b, c (Å)132.461, 162.908, 139.325
Angle α, β, γ (deg.)90.000, 107.320, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 6 molecules ABTCDE

#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.: 3 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / 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 natural source
Source: (natural) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / References: UniProt: P60281, DNA-directed RNA polymerase
#3: 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 natural source
Source: (natural) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / References: UniProt: A0QS66, DNA-directed RNA polymerase
#4: 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 natural source
Source: (natural) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / References: UniProt: A0QWT1, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules FJ

#5: Protein RNA polymerase sigma factor SigA


Mass: 51573.551 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / 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 (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: rbpA, MSMEG_3858, MSMEI_3768
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0QZ11

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Protein/peptide , 1 types, 1 molecules G

#6: Protein/peptide unknown


Mass: 1464.797 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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DNA chain , 2 types, 2 molecules OP

#8: DNA chain DNA (31-MER)


Mass: 9565.193 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#9: DNA chain DNA (26-MER)


Mass: 7930.155 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 5 types, 67 molecules

#10: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: SO4 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C2H6O2 / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical ChemComp-SRN / SORANGICIN A


Mass: 807.020 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H66O11 / Feature type: SUBJECT OF INVESTIGATION
#13: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#14: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 43 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.96 Å3/Da / Density % sol: 58.5 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop
Details: 0.1 M Bis-Tris, pH 6.0, 0.2 M lithium sulfate, 20% w/v PEG3350, 2.5% v/v ethylene glycol, 1% v/v DMSO

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Data collection

DiffractionMean temperature: 80 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Apr 24, 2016
RadiationMonochromator: cryo-cooled double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 3.1→55 Å / Num. obs: 98175 / % possible obs: 97.7 % / Redundancy: 10.5 % / Biso Wilson estimate: 94.8 Å2 / Rmerge(I) obs: 0.162 / Rpim(I) all: 0.051 / Rrim(I) all: 0.17 / Χ2: 1.099 / Net I/σ(I): 5.6 / Num. measured all: 1026574
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.1-3.217.12.04584350.5290.7622.1910.54483.8
3.21-3.348.21.25493590.6610.4371.3330.56593.9
3.34-3.4910.10.83999740.8570.2710.8830.53699.7
3.49-3.6811.10.599100000.9270.1880.6280.591100
3.68-3.9111.10.46100120.9620.1450.4830.744100
3.91-4.2110.80.279100130.9830.0890.2930.907100
4.21-4.6311.90.173100590.9930.0520.181.211100
4.63-5.311.10.14100330.9940.0430.1461.329100
5.3-6.6811.70.111100820.9960.0340.1171.501100
6.68-5510.70.053102080.9990.0170.0552.528100

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Processing

Software
NameVersionClassification
PHENIXv0refinement
HKL-2000data scaling
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 5TW1

5tw1


Resolution: 3.112→54.665 Å / SU ML: 0.49 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 31.33
RfactorNum. reflection% reflection
Rfree0.258 1997 2.04 %
Rwork0.2159 --
obs0.2168 97815 96.72 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 410.6 Å2 / Biso mean: 105.386 Å2 / Biso min: 29.81 Å2
Refinement stepCycle: final / Resolution: 3.112→54.665 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms25204 1159 200 57 26620
Biso mean--117.41 76.26 -
Num. residues----3387
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00327047
X-RAY DIFFRACTIONf_angle_d0.57136930
X-RAY DIFFRACTIONf_chiral_restr0.0414276
X-RAY DIFFRACTIONf_plane_restr0.0044642
X-RAY DIFFRACTIONf_dihedral_angle_d14.10416188
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.1121-3.190.39491100.387490270
3.19-3.27620.36391130.3252628189
3.2762-3.37260.32691590.3026673596
3.3726-3.48140.32291310.29447069100
3.4814-3.60580.3111600.26727030100
3.6058-3.75020.32211400.26057090100
3.7502-3.92080.33391530.24547017100
3.9208-4.12740.29721380.22867099100
4.1274-4.38590.24321500.20727045100
4.3859-4.72440.22721500.18777057100
4.7244-5.19950.22891440.19867092100
5.1995-5.95110.24671520.20797074100
5.9511-7.49470.28331410.21267116100
7.4947-54.660.18921560.16197211100

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