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- EMDB-22578: Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/So... -

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Basic information

Entry
Database: EMDB / ID: EMD-22578
TitleMycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/Sor/AP3 - PreRP2 class
Map data
Sample
  • Complex: Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/Sor/AP3 - PreRP2 class
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.38 Å
AuthorsLilic M / Chen J / Darst SA / Campbell EA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2-R01 GM114450 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
DepositionSep 2, 2020-
Header (metadata) releaseOct 21, 2020-
Map releaseOct 21, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22578.png

Downloads & links

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Map

FileDownload / File: emd_22578.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.45
Minimum - Maximum-0.45649862 - 1.3609884
Average (Standard dev.)0.0076125446 (±0.06548374)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.4561.3610.008

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Supplemental data

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Sample components

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Entire : Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/So...

EntireName: Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/Sor/AP3 - PreRP2 class
Components
  • Complex: Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/Sor/AP3 - PreRP2 class

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Supramolecule #1: Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/So...

SupramoleculeName: Mycobacterium tuberculosis BetaS456L-RNAP holoenzyme/RbpA/CarD/Sor/AP3 - PreRP2 class
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 71.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.38 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 27852

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