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- PDB-6iol: Cryo-EM structure of multidrug efflux pump MexAB-OprM (60 degree ... -

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Basic information

Entry
Database: PDB / ID: 6iol
TitleCryo-EM structure of multidrug efflux pump MexAB-OprM (60 degree state)
Components
  • Multidrug resistance protein MexA
  • Multidrug resistance protein MexB
  • Outer membrane protein OprM
KeywordsMEMBRANE PROTEIN / Multidrug resistance / efflux / complex
Function / homology
Function and homology information


efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / transmembrane transporter activity / cell outer membrane / protein homooligomerization / transmembrane transport / response to antibiotic / identical protein binding / membrane / plasma membrane
Similarity search - Function
Outer membrane efflux proteins (OEP) / Outer membrane efflux proteins (OEP) / : / Outer membrane efflux proteins (OEP) / Outer membrane efflux proteins (OEP) / RND efflux system, outer membrane lipoprotein, NodT / : / Cation efflux system protein CusB, domain 1 / Cation efflux system protein CusB domain 1 / RND efflux pump, membrane fusion protein ...Outer membrane efflux proteins (OEP) / Outer membrane efflux proteins (OEP) / : / Outer membrane efflux proteins (OEP) / Outer membrane efflux proteins (OEP) / RND efflux system, outer membrane lipoprotein, NodT / : / Cation efflux system protein CusB, domain 1 / Cation efflux system protein CusB domain 1 / RND efflux pump, membrane fusion protein / RND efflux pump, membrane fusion protein, barrel-sandwich domain / Barrel-sandwich domain of CusB or HlyD membrane-fusion / Outer membrane efflux protein / Outer membrane efflux protein / Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / Single Sheet / Prokaryotic membrane lipoprotein lipid attachment site profile. / Up-down Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Multidrug resistance protein MexB / Multidrug resistance protein MexA / Outer membrane protein OprM
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Pseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsTsutsumi, K. / Yonehara, R. / Nakagawa, A. / Yamashita, E.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of ScienceJP25291014 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)JP22121006 Japan
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Nat Commun / Year: 2019
Title: Structures of the wild-type MexAB-OprM tripartite pump reveal its complex formation and drug efflux mechanism.
Authors: Kenta Tsutsumi / Ryo Yonehara / Etsuko Ishizaka-Ikeda / Naoyuki Miyazaki / Shintaro Maeda / Kenji Iwasaki / Atsushi Nakagawa / Eiki Yamashita /
Abstract: In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the ...In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB-OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB-OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux.
History
DepositionOct 30, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 3, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
I: Multidrug resistance protein MexA
J: Multidrug resistance protein MexA
K: Multidrug resistance protein MexA
L: Multidrug resistance protein MexA
M: Multidrug resistance protein MexA
N: Multidrug resistance protein MexA
A: Outer membrane protein OprM
B: Outer membrane protein OprM
C: Outer membrane protein OprM
F: Multidrug resistance protein MexB
G: Multidrug resistance protein MexB
E: Multidrug resistance protein MexB


Theoretical massNumber of molelcules
Total (without water)729,79612
Polymers729,79612
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area78560 Å2
ΔGint-310 kcal/mol
Surface area253060 Å2

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Components

#1: Protein
Multidrug resistance protein MexA


Mass: 38789.695 Da / Num. of mol.: 6 / Fragment: UNP residues 25-383
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Strain: PAO1 / Gene: mexA / Production host: Escherichia coli (E. coli) / References: UniProt: P52477
#2: Protein Outer membrane protein OprM


Mass: 51737.605 Da / Num. of mol.: 3 / Fragment: UNP residues 18-485
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1 / Gene: oprM / Production host: Escherichia coli (E. coli) / References: UniProt: Q51487
#3: Protein Multidrug resistance protein MexB / Multidrug-efflux transporter MexB


Mass: 113948.273 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1 / Gene: mexB / Production host: Escherichia coli (E. coli) / References: UniProt: P52002

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: membrane protein complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.73 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer componentConc.: 50 mM / Name: HEPES / Formula: C8H18N2O4S
SpecimenConc.: 9.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1250 nm / Cs: 0.1 mm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 95 K / Temperature (min): 90 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8722
EM imaging opticsSpherical aberration corrector: CEOS Cs-corrector
Image scansSampling size: 14 µm / Width: 4080 / Height: 4080 / Movie frames/image: 32 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
4Gctf1.06CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 535948
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42338 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00549906
ELECTRON MICROSCOPYf_angle_d0.96167805
ELECTRON MICROSCOPYf_dihedral_angle_d11.20930194
ELECTRON MICROSCOPYf_chiral_restr0.0577920
ELECTRON MICROSCOPYf_plane_restr0.0078865

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