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Yorodumi- PDB-7szj: Cryo-EM structure of Rifamycin bound to E. coli RNAP and rrnBP1 p... -
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-Basic information
Entry | Database: PDB / ID: 7szj | ||||||
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Title | Cryo-EM structure of Rifamycin bound to E. coli RNAP and rrnBP1 promoter complex | ||||||
Components |
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Keywords | TRANSFERASE/DNA/ANTIBIOTIC / Transcription / antibiotics / Rifamycin / TRANSFERASE-DNA-ANTIBIOTIC complex | ||||||
Function / homology | Function and homology information sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / 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 / negative regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | Escherichia coli K-12 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å | ||||||
Authors | Shin, Y. / Murakami, K.S. | ||||||
Funding support | United States, 1items
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Citation | Journal: ACS Infect Dis / Year: 2022 Title: Optimization of Benzoxazinorifamycins to Improve RNA Polymerase Inhibition and Treatment of Tuberculosis. Authors: Walajapet Rajeswaran / Shireen R Ashkar / Pil H Lee / Larisa Yeomans / Yeonoh Shin / Scott G Franzblau / Katsuhiko S Murakami / Hollis D Showalter / George A Garcia / Abstract: Rifampin (RMP), a very potent inhibitor of the (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from ...Rifampin (RMP), a very potent inhibitor of the (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIF) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIF) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue () that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (∼30-fold > RMP) activity against RIF MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, has a 7 day MBC that is ∼10-fold more potent than RMP. These results suggest that may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of ∼2 g of at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7szj.cif.gz | 717.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7szj.ent.gz | 555.5 KB | Display | PDB format |
PDBx/mmJSON format | 7szj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7szj_validation.pdf.gz | 1015.2 KB | Display | wwPDB validaton report |
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Full document | 7szj_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7szj_validation.xml.gz | 114.1 KB | Display | |
Data in CIF | 7szj_validation.cif.gz | 171.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sz/7szj ftp://data.pdbj.org/pub/pdb/validation_reports/sz/7szj | HTTPS FTP |
-Related structure data
Related structure data | 25570MC 7szkC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#1: Protein | 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 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase #2: Protein | | 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 Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8V2, DNA-directed RNA polymerase #3: Protein | | 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 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase #4: Protein | | 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 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase |
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-Protein , 1 types, 1 molecules F
#5: Protein | Mass: 70352.242 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P00579 |
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-DNA chain , 2 types, 2 molecules XY
#6: DNA chain | Mass: 19552.533 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria) |
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#7: DNA chain | Mass: 19903.734 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria) |
-Non-polymers , 3 types, 4 molecules
#8: Chemical | ChemComp-RFP / |
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#9: Chemical | ChemComp-MG / |
#10: Chemical |
-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Rifamycin bound to E. coli RNAP and rrnBP1 promoter complex Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli K-12 (bacteria) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: OTHER |
Image recording | Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3301 |
-Processing
Software |
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EM software | Name: cryoSPARC / Version: 3.0.0 / Category: 3D reconstruction | ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251427 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 50.23 Å2 | ||||||||||||||||||||||||
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