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- PDB-6ta5: OprM-MexA complex from the MexAB-OprM Pseudomonas aeruginosa whol... -

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Basic information

Entry
Database: PDB / ID: 6ta5
TitleOprM-MexA complex from the MexAB-OprM Pseudomonas aeruginosa whole assembly reconstituted in nanodiscs
Components
  • Efflux pump membrane transporter
  • MexA family multidrug efflux RND transporter periplasmic adaptor subunit
  • Outer membrane protein OprM
KeywordsANTIMICROBIAL PROTEIN / transporter / efflux / proton motive force / bacterial resistance
Function / homology
Function and homology information


response to chemical / xenobiotic transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transporter activity / cell outer membrane / protein homooligomerization / transmembrane transport / response to antibiotic / membrane ...response to chemical / xenobiotic transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transporter activity / cell outer membrane / protein homooligomerization / transmembrane transport / response to antibiotic / membrane / identical protein binding / plasma membrane
Similarity search - Function
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 ...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 / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Efflux pump membrane transporter / MexA family multidrug efflux RND transporter periplasmic adaptor subunit / Multidrug resistance protein MexB / Multidrug resistance protein MexA / Outer membrane protein OprM
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGlavier, M. / Schoehn, G. / Taveau, J.C. / Phan, G. / Daury, L. / Lambert, O. / Broutin, I.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research AgencyANR-17-CE11-0028-02 France
CitationJournal: Nat Commun / Year: 2020
Title: Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.
Authors: Marie Glavier / Dhenesh Puvanendran / Dimitri Salvador / Marion Decossas / Gilles Phan / Cyril Garnier / Elisa Frezza / Quentin Cece / Guy Schoehn / Martin Picard / Jean-Christophe Taveau / ...Authors: Marie Glavier / Dhenesh Puvanendran / Dimitri Salvador / Marion Decossas / Gilles Phan / Cyril Garnier / Elisa Frezza / Quentin Cece / Guy Schoehn / Martin Picard / Jean-Christophe Taveau / Laetitia Daury / Isabelle Broutin / Olivier Lambert /
Abstract: The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB ...The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.
History
DepositionOct 29, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 2020Group: 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Outer membrane protein OprM
B: Outer membrane protein OprM
C: Outer membrane protein OprM
D: MexA family multidrug efflux RND transporter periplasmic adaptor subunit
E: MexA family multidrug efflux RND transporter periplasmic adaptor subunit
F: MexA family multidrug efflux RND transporter periplasmic adaptor subunit
G: MexA family multidrug efflux RND transporter periplasmic adaptor subunit
H: MexA family multidrug efflux RND transporter periplasmic adaptor subunit
I: MexA family multidrug efflux RND transporter periplasmic adaptor subunit
J: Efflux pump membrane transporter
K: Efflux pump membrane transporter
L: Efflux pump membrane transporter


Theoretical massNumber of molelcules
Total (without water)733,10512
Polymers733,10512
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area74750 Å2
ΔGint-302 kcal/mol
Surface area264840 Å2
MethodPISA

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Components

#1: Protein Outer membrane protein OprM


Mass: 51737.605 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: trimer of the outer membrane factor OprM / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: oprM, oprK, PA0427 / Production host: Escherichia coli (E. coli) / References: UniProt: Q51487
#2: Protein
MexA family multidrug efflux RND transporter periplasmic adaptor subunit


Mass: 39462.387 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: trimer of dimers of the membrane fusion protein MexA
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: mexA, CAZ10_35280, CGU42_31245, DT376_09430, DZ934_11470, DZ962_14980, E4V10_16060, EFK68_29775, EQH76_08910, IPC3_09880, IPC669_14605
Production host: Escherichia coli (E. coli) / References: UniProt: A0A2V3GTR8, UniProt: P52477*PLUS
#3: Protein Efflux pump membrane transporter


Mass: 113706.008 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: trimer of the RND transporter MexB / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: mexB, mexB_1, C0044_03030, DZ934_11475, E4V10_16065, IPC3_09885, IPC669_14600, PAERUG_E15_London_28_01_14_02845, PAMH19_0483, RW109_RW109_01030
Production host: Escherichia coli (E. coli) / References: UniProt: A0A069Q9M6, UniProt: P52002*PLUS

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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: MexAB-OprM in nanodisc / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17_3644refinement
PHENIX1.17_3644refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimera1.10.2model fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
19PHENIX1.14model refinement1.17 to create the .cif
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 364914
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35819 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16TA6

6ta6

D1
26TA6

6ta6

E1
36TA6

6ta6

F1
46TA6

6ta6

G1
56TA6

6ta6

H1
66TA6

6ta6

I1
76TA6

6ta6

J1
86TA6

6ta6

K1
96TA6

6ta6

L1
103D5K

3d5k

A1
113D5K

3d5k

B1
123D5K

3d5k

C1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 104.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00550403
ELECTRON MICROSCOPYf_angle_d0.933368493
ELECTRON MICROSCOPYf_chiral_restr0.0567983
ELECTRON MICROSCOPYf_plane_restr0.00678973
ELECTRON MICROSCOPYf_dihedral_angle_d16.381118519

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