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- PDB-6fbv: Single particle cryo em structure of Mycobacterium tuberculosis R... -
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Basic information
Entry | Database: PDB / ID: 6fbv | ||||||
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Title | Single particle cryo em structure of Mycobacterium tuberculosis RNA polymerase in complex with Fidaxomicin | ||||||
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![]() | TRANSCRIPTION / Lipiarmycin / RNA pol / RNAP / inhibitor / drug / Clostridium difficile / ANTIBIOTIC / Tiacumicin B / CCDC 114782 | ||||||
Function / homology | ![]() response to water / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity ...response to water / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / 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 / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å | ||||||
![]() | Das, K. / Lin, W. / Ebright, E. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3). Authors: Wei Lin / Kalyan Das / David Degen / Abhishek Mazumder / Diego Duchi / Dongye Wang / Yon W Ebright / Richard Y Ebright / Elena Sineva / Matthew Gigliotti / Aashish Srivastava / Sukhendu ...Authors: Wei Lin / Kalyan Das / David Degen / Abhishek Mazumder / Diego Duchi / Dongye Wang / Yon W Ebright / Richard Y Ebright / Elena Sineva / Matthew Gigliotti / Aashish Srivastava / Sukhendu Mandal / Yi Jiang / Yu Liu / Ruiheng Yin / Zhening Zhang / Edward T Eng / Dennis Thomas / Stefano Donadio / Haibo Zhang / Changsheng Zhang / Achillefs N Kapanidis / Richard H Ebright / ![]() ![]() ![]() ![]() ![]() Abstract: Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial ...Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives. | ||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 648.7 KB | Display | ![]() |
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PDB format | ![]() | 514.3 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 4230MC ![]() 6asgC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#1: Protein | Mass: 37745.328 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: rpoA, Rv3457c, MTCY13E12.10c / Production host: ![]() ![]() #2: Protein | | Mass: 130018.828 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: rpoB, Rv0667, MTCI376.08c / Production host: ![]() ![]() #3: Protein | | Mass: 146968.969 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: rpoC, Rv0668, MTCI376.07c / Production host: ![]() ![]() #4: Protein | | Mass: 11851.140 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: rpoZ, Rv1390, MTCY21B4.07 / Production host: ![]() ![]() |
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-Protein , 1 types, 1 molecules F
#5: Protein | Mass: 57877.160 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: sigA, mysA, rpoD, rpoV, Rv2703, MTCY05A6.24 / Production host: ![]() ![]() |
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-Non-polymers , 4 types, 7 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/FI8.gif)
![](data/chem/img/HOH.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/FI8.gif)
![](data/chem/img/HOH.gif)
#6: Chemical | #7: Chemical | ChemComp-MG / | #8: Chemical | ChemComp-FI8 / | #9: Water | ChemComp-HOH / | |
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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 |
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Sample preparation
Component | Name: structure of Mycobacterium tuberculosis RNA polymerase in complex with Fidaxomicin Type: COMPLEX / Details: RNA polymerase inhibitor complex / Entity ID: #1-#5 / Source: RECOMBINANT |
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Molecular weight | Units: MEGADALTONS |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 Details: 3.5 microliter 1 microM Mtb RNAP-Lpm and 50 microMolar Lpm in 20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 5 mM MgCl2, 5 mM dithiothreitol, and 0.1% n-octyl-beta-D-glucopyranoside |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. |
Vitrification | Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 291 K |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: -2 nm / Nominal defocus min: -1 nm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.2 sec. / Electron dose: 1.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2458 Details: Movies were recorded at 200 ms/frame for 10s (50 frames total), resulting in a total radiation dose of 72.05 electrons/A**2 per movie Defocus range was varied between 1.0 - 2.0 micrometer. |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-35 |
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Processing
Software | Name: PHENIX / Version: (1.11.1_2575: ???) / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 819506 / Details: Autopick | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68895 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 95.3 / Protocol: OTHER / Target criteria: Cross-correlation coefficient | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3.52→3.52 Å / SU ML: 0.81 / σ(F): 0.06 / Phase error: 40.48 / Stereochemistry target values: MLHL
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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