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- EMDB-10372: CryoEM structure of MexAB-OprM in nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-10372
TitleCryoEM structure of MexAB-OprM in nanodisc
Map dataCryoEM structure of Pseudomonas aeruginosa MexAB-OprM complex stabilized in nanodisc
Sample
  • Complex: Tripartite complex MexAB-OprM
    • Protein or peptide: mexB
    • Protein or peptide: mexA
    • Protein or peptide: OprM
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGlavier M / Puvanendran D / Salvador D / Decossas M / Phan G / Garnier C / Frezza E / Cece Q / Schoehn G / Picard M ...Glavier M / Puvanendran D / Salvador D / Decossas M / Phan G / Garnier C / Frezza E / Cece Q / Schoehn G / Picard M / Taveau J-C / Daury L / Broutin I / Lambert O
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyANR-17CE11-0028 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 9, 2019-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ta5
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10372.png

Downloads & links

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Map

FileDownload / File: emd_10372.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of Pseudomonas aeruginosa MexAB-OprM complex stabilized in nanodisc
Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.09764242 - 0.17524983
Average (Standard dev.)-7.3432925e-06 (±0.0052246875)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 696.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z696.320696.320696.320
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0980.175-0.000

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Supplemental data

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Sample components

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Entire : Tripartite complex MexAB-OprM

EntireName: Tripartite complex MexAB-OprM
Components
  • Complex: Tripartite complex MexAB-OprM
    • Protein or peptide: mexB
    • Protein or peptide: mexA
    • Protein or peptide: OprM

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Supramolecule #1: Tripartite complex MexAB-OprM

SupramoleculeName: Tripartite complex MexAB-OprM / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: mexB

MacromoleculeName: mexB / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MSKFFIDRPI FAWVIALVIM LAGGLSILSL PVNQYPAIAP PAIAVQVSYP GASAETVQDT VVQVIEQQM NGIDNLRYIS SESNSDGSMT ITVTFEQGTD PDIAQVQVQN KLQLATPLLP Q EVQRQGIR VTKAVKNFLM VVGVVSTDGS MTKEDLSNYI VSNIQDPLSR ...String:
MSKFFIDRPI FAWVIALVIM LAGGLSILSL PVNQYPAIAP PAIAVQVSYP GASAETVQDT VVQVIEQQM NGIDNLRYIS SESNSDGSMT ITVTFEQGTD PDIAQVQVQN KLQLATPLLP Q EVQRQGIR VTKAVKNFLM VVGVVSTDGS MTKEDLSNYI VSNIQDPLSR TKGVGDFQVF GS QYSMRIW LDPAKLNSYQ LTPGDVSSAI QAQNVQISSG QLGGLPAVKG QQLNATIIGK TRL QTAEQF ENILLKVNPD GSQVRLKDVA DVGLGGQDYS INAQFNGSPA SGIAIKLATG ANAL DTAKA IRQTIANLEP FMPQGMKVVY PYDTTPVVSA SIHEVVKTLG EAILLVFLVM YLFLQ NFRA TLIPTIAVPV VLLGTFGVLA AFGFSINTLT MFGMVLAIGL LVDDAIVVVE NVERVM AEE GLSPREAARK SMGQIQGALV GIAMVLSAVF LPMAFFGGST GVIYRQFSIT IVSAMAL SV IVALILTPAL CATMLKPIEK GDHGEHKGGF FGWFNRMFLS TTHGYERGVA SILKHRAP Y LLIYVVIVAG MIWMFTRIPT AFLPDEDQGV LFAQVQTPPG SSAERTQVVV DSMREYLLE KESSSVSSVF TVTGFNFAGR GQSSGMAFIM LKPWEERPGG ENSVFELAKR AQMHFFSFKD AMVFAFAPP SVLELGNATG FDLFLQDQAG VGHEVLLQAR NKFLMLAAQN PALQRVRPNG M SDEPQYKL EIDDEKASAL GVSLADINST VSIAWGSSYV NDFIDRGRVK RVYLQGRPDA RM NPDDLSK WYVRNDKGEM VPFNAFATGK WEYGSPKLER YNGVPAMEIL GEPAPGLSSG DAM AAVEEI VKQLPKGVGY SWTGLSYEER LSGSQAPALY ALSLLVVFLC LAALYESWSI PFSV MLVVP LGVIGALLAT SMRGLSNDVF FQVGLLTTIG LSAKNAILIV EFAKELHEQG KGIVE AAIE ACRMRLRPIV MTSLAFILGV VPLAISTGAG SGSQHAIGTG VIGGMVTATV LAIFWV PLF YVAVSTLFKD EASKQQASVE KGQ

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Macromolecule #2: mexA

MacromoleculeName: mexA / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
SequenceString: MQRTPAMRVL VPALLVAISA LSGCGKSEAP PPAQTPEVGI VTLEAQTVTL NTELPGRTNA FRIAEVRPQ VNGIILKRLF KEGSDVKAGQ QLYQIDPATY EADYQSAQAN LASTQEQAQR Y KLLVADQA VSKQQYADAN AAYLQSKAAV EQARINLRYT KVLSPISGRI ...String:
MQRTPAMRVL VPALLVAISA LSGCGKSEAP PPAQTPEVGI VTLEAQTVTL NTELPGRTNA FRIAEVRPQ VNGIILKRLF KEGSDVKAGQ QLYQIDPATY EADYQSAQAN LASTQEQAQR Y KLLVADQA VSKQQYADAN AAYLQSKAAV EQARINLRYT KVLSPISGRI GRSAVTEGAL VT NGQANAM ATVQQLDPIY VDVTQPSTAL LRLRRELASG QLERAGDNAA KVSLKLEDGS QYP LEGRLE FSEVSVDEGT GSVTIRAVFP NPNNELLPGM FVHAQLQEGV KQKAILAPQQ GVTR DLKGQ ATALVVNAQN KVELRVIKAD RVIGDKWLVT EGLNAGDKII TEGLQFVQPG VEVKT VPAK NVASAQKADA APAKTDSKG

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Macromolecule #3: OprM

MacromoleculeName: OprM / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
SequenceString: MKRSFLSLAV AAVVLSGCSL IPDYQRPEAP VAAAYPQGQA YGQNTGAAAV PAADIGWREF FRDPQLQQL IGVALENNRD LRVAALNVEA FRAQYRIQRA DLFPRIGVDG SGTRQRLPGD L STTGSPAI SSQYGVTLGT TAWELDLFGR LRSLRDQALE QYLATEQAQR ...String:
MKRSFLSLAV AAVVLSGCSL IPDYQRPEAP VAAAYPQGQA YGQNTGAAAV PAADIGWREF FRDPQLQQL IGVALENNRD LRVAALNVEA FRAQYRIQRA DLFPRIGVDG SGTRQRLPGD L STTGSPAI SSQYGVTLGT TAWELDLFGR LRSLRDQALE QYLATEQAQR SAQTTLVASV AT AYLTLKA DQAQLQLTKD TLGTYQKSFD LTQRSYDVGV ASALDLRQAQ TAVEGARATL AQY TRLVAQ DQNALVLLLG SGIPANLPQG LGLDQTLLTE VPAGLPSDLL QRRPDILEAE HQLM AANAS IGAARAAFFP SISLTANAGT MSRQLSGLFD AGSGSWLFQP SINLPIFTAG SLRAS LDYA KIQKDINVAQ YEKAIQTAFQ EVADGLAARG TFTEQLQAQR DLVKASDEYY QLADKR YRT GVDNYLTLLD AQRSLFTAQQ QLITDRLNQL TSEVNLYKAL GGGWNQQTVT QQQTAKK ED PQA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 364914
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 35819
FSC plot (resolution estimation)

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