+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6317 | |||||||||
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Title | Three-dimensional structure of AcrAB-TolC complex | |||||||||
Map data | Reconstruction of negatively stained AcrB-transmembrane linker-AcrA-AcrA and TolC complex, which supports 3:6:3 (AcrB:AcrA:TolC) assembly stoichiometry and the direct interaction between AcrA and TolC | |||||||||
Sample |
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Keywords | multidrug efflux pump / multidrug resistance / resistance-nodulation-division family | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 26.0 Å | |||||||||
Authors | Kim J-S / Jeong H / Song S / Kim H-Y / Lee K / Hyun J / Ha N-C | |||||||||
Citation | Journal: Mol Cells / Year: 2015 Title: Structure of the tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode. Authors: Jin-Sik Kim / Hyeongseop Jeong / Saemee Song / Hye-Yeon Kim / Kangseok Lee / Jaekyung Hyun / Nam-Chul Ha / Abstract: Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation ...Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6317.map.gz | 9.6 MB | EMDB map data format | |
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Header (meta data) | emd-6317-v30.xml emd-6317.xml | 10.2 KB 10.2 KB | Display Display | EMDB header |
Images | 400_6317.gif 80_6317.gif | 24.8 KB 2.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6317 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6317 | HTTPS FTP |
-Validation report
Summary document | emd_6317_validation.pdf.gz | 78.7 KB | Display | EMDB validaton report |
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Full document | emd_6317_full_validation.pdf.gz | 77.8 KB | Display | |
Data in XML | emd_6317_validation.xml.gz | 495 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6317 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6317 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6317.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of negatively stained AcrB-transmembrane linker-AcrA-AcrA and TolC complex, which supports 3:6:3 (AcrB:AcrA:TolC) assembly stoichiometry and the direct interaction between AcrA and TolC | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : AcrB-TM#5-AcrA-AcrA and TolC complex
Entire | Name: AcrB-TM#5-AcrA-AcrA and TolC complex |
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Components |
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-Supramolecule #1000: AcrB-TM#5-AcrA-AcrA and TolC complex
Supramolecule | Name: AcrB-TM#5-AcrA-AcrA and TolC complex / type: sample / ID: 1000 / Oligomeric state: AcrB trimer, AcrA hexamer and TolC trimer / Number unique components: 1 |
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Molecular weight | Theoretical: 700 KDa |
-Macromolecule #1: Multidrug efflux pump AcrAB-TolC
Macromolecule | Name: Multidrug efflux pump AcrAB-TolC / type: protein_or_peptide / ID: 1 Details: AcrAB fusion protein was generated by fusing functional AcrA dimer to the C-terminus of AcrB with a short transmembrane linker, and TolC was bound to the fusion protein in order to generate ...Details: AcrAB fusion protein was generated by fusing functional AcrA dimer to the C-terminus of AcrB with a short transmembrane linker, and TolC was bound to the fusion protein in order to generate the complete tripartite complex Number of copies: 12 / Oligomeric state: Dodecamer (3 x AcrB, 6 x AcrA, 3 x TolC) / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 700 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET22b |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 7.5 / Details: 20mM HEPES, 150mM NaCl, 0.02% DDM, 10% glycerol |
Staining | Type: NEGATIVE Details: Grid was stained with 0.75% uranyl formate for 60 seconds, followed by blotting of excess solution using a piece of filter paper |
Grid | Details: 300-mesh copper EM-grid with covered with thin carbon film, glow discharged in air |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI SPIRIT |
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Date | Apr 20, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 100 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 66667 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 52000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | Model: Tecnai Spirit / Image courtesy: FEI Company |
-Image processing
Details | Particles were manually selected and boxed in 256 x 256 pixel boxes using e2boxer.py. Boxed particles were used to estimate and correct for CTF using e2ctf.py. Initial model from selected class averages was generated using e2initialmodel.py, and the 3D refinement was performed using e2refine_easy.py. |
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CTF correction | Details: CTF correction to each particle |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 2591 |