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- PDB-6t7s: MexB structure solved by cryo-EM in nanodisc in absence of its pr... -

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Basic information

Entry
Database: PDB / ID: 6t7s
TitleMexB structure solved by cryo-EM in nanodisc in absence of its protein partners
ComponentsEfflux pump membrane transporter
KeywordsANTIMICROBIAL PROTEIN / transporter / efflux / proton motive force / bacterial resistance
Function / homology
Function and homology information


xenobiotic transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transport / response to antibiotic / plasma membrane
Similarity search - Function
Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Efflux pump membrane transporter / Multidrug resistance protein MexB
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 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 23, 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
J: Efflux pump membrane transporter
K: Efflux pump membrane transporter
L: Efflux pump membrane transporter


Theoretical massNumber of molelcules
Total (without water)341,1183
Polymers341,1183
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18960 Å2
ΔGint-93 kcal/mol
Surface area124600 Å2
MethodPISA

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Components

#1: Protein Efflux pump membrane transporter


Mass: 113706.008 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
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: MexB / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
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

EM software
IDNameCategory
1RELIONparticle selection
4GctfCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41604 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 239.16 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003723913
ELECTRON MICROSCOPYf_angle_d0.921932499
ELECTRON MICROSCOPYf_chiral_restr0.05593792
ELECTRON MICROSCOPYf_plane_restr0.0074173
ELECTRON MICROSCOPYf_dihedral_angle_d15.65438637

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