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- EMDB-9696: Cryo-EM structure of multidrug efflux pump MexAB-OprM (60 degree ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9696
TitleCryo-EM structure of multidrug efflux pump MexAB-OprM (60 degree state)
Map dataSharpened with B-factor sharpening.
Sample
  • Complex: membrane protein complexBiological membrane
    • Protein or peptide: Multidrug resistance protein MexAMultiple drug resistance
  • Protein or peptide: Outer membrane protein OprM
  • Protein or peptide: Multidrug resistance protein MexBMultiple drug resistance
KeywordsMultidrug resistance / efflux / complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


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
Multidrug resistance protein MexB / Multidrug resistance protein MexA / Outer membrane protein OprM
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Pseudomonas aeruginosa PAO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsTsutsumi K / Yonehara R
Funding support Japan, 3 items
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, 2018-
Header (metadata) releaseApr 3, 2019-
Map releaseApr 3, 2019-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6iol
  • Surface level: 10
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9696.png

Downloads & links

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Map

FileDownload / File: emd_9696.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened with B-factor sharpening.
Voxel sizeX=Y=Z: 0.875 Å
Density
Contour LevelBy AUTHOR: 3.65 / Movie #1: 10
Minimum - Maximum-76.440055999999998 - 99.154754999999994
Average (Standard dev.)0.022707604 (±2.5606613)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 350.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8750.8750.875
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z350.000350.000350.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-76.44099.1550.023

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Supplemental data

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Half map: #1

Fileemd_9696_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_9696_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : membrane protein complex

EntireName: membrane protein complexBiological membrane
Components
  • Complex: membrane protein complexBiological membrane
    • Protein or peptide: Multidrug resistance protein MexAMultiple drug resistance
  • Protein or peptide: Outer membrane protein OprM
  • Protein or peptide: Multidrug resistance protein MexBMultiple drug resistance

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Supramolecule #1: membrane protein complex

SupramoleculeName: membrane protein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 730 KDa

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Macromolecule #1: Multidrug resistance protein MexA

MacromoleculeName: Multidrug resistance protein MexA / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: PAO1
Molecular weightTheoretical: 38.789695 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMGKSEAPP PAQTPEVGIV TLEAQTVTLN TELPGRTNAF RIAEVRPQVN GIILKRLFKE GSDVKAGQQL YQIDPATYEA DYQSAQANL ASTQEQAQRY KLLVADQAVS KQQYADANAA YLQSKAAVEQ ARINLRYTKV LSPISGRIGR SAVTEGALVT N GQANAMAT ...String:
GAMGKSEAPP PAQTPEVGIV TLEAQTVTLN TELPGRTNAF RIAEVRPQVN GIILKRLFKE GSDVKAGQQL YQIDPATYEA DYQSAQANL ASTQEQAQRY KLLVADQAVS KQQYADANAA YLQSKAAVEQ ARINLRYTKV LSPISGRIGR SAVTEGALVT N GQANAMAT VQQLDPIYVD VTQPSTALLR LRRELASGQL ERAGDNAAKV SLKLEDGSQY PLEGRLEFSE VSVDEGTGSV TI RAVFPNP NNELLPGMFV HAQLQEGVKQ KAILAPQQGV TRDLKGQATA LVVNAQNKVE LRVIKADRVI GDKWLVTEGL NAG DKIITE GLQFVQPGVE VKTVPAKNVA SAQKADAAPA KTDSKG

UniProtKB: Multidrug resistance protein MexA

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Macromolecule #2: Outer membrane protein OprM

MacromoleculeName: Outer membrane protein OprM / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1
Molecular weightTheoretical: 51.737605 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: CSLIPDYQRP EAPVAAAYPQ GQAYGQNTGA AAVPAADIGW REFFRDPQLQ QLIGVALENN RDLRVAALNV EAFRAQYRIQ RADLFPRIG VDGSGTRQRL PGDLSTTGSP AISSQYGVTL GTTAWELDLF GRLRSLRDQA LEQYLATEQA QRSAQTTLVA S VATAYLTL ...String:
CSLIPDYQRP EAPVAAAYPQ GQAYGQNTGA AAVPAADIGW REFFRDPQLQ QLIGVALENN RDLRVAALNV EAFRAQYRIQ RADLFPRIG VDGSGTRQRL PGDLSTTGSP AISSQYGVTL GTTAWELDLF GRLRSLRDQA LEQYLATEQA QRSAQTTLVA S VATAYLTL KADQAQLQLT KDTLGTYQKS FDLTQRSYDV GVASALDLRQ AQTAVEGARA TLAQYTRLVA QDQNALVLLL GS GIPANLP QGLGLDQTLL TEVPAGLPSD LLQRRPDILE AEHQLMAANA SIGAARAAFF PSISLTANAG TMSRQLSGLF DAG SGSWLF QPSINLPIFT AGSLRASLDY AKIQKDINVA QYEKAIQTAF QEVADGLAAR GTFTEQLQAQ RDLVKASDEY YQLA DKRYR TGVDNYLTLL DAQRSLFTAQ QQLITDRLNQ LTSEVNLYKA LGGGWNQQTV TQQQTAKKED PQAHHHHHH

UniProtKB: Outer membrane protein OprM

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Macromolecule #3: Multidrug resistance protein MexB

MacromoleculeName: Multidrug resistance protein MexB / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1
Molecular weightTheoretical: 113.948273 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKFFIDRPI FAWVIALVIM LAGGLSILSL PVNQYPAIAP PAIAVQVSYP GASAETVQDT VVQVIEQQMN GIDNLRYISS ESNSDGSMT ITVTFEQGTD PDIAQVQVQN KLQLATPLLP QEVQRQGIRV TKAVKNFLMV VGVVSTDGSM TKEDLSNYIV S NIQDPLSR ...String:
MSKFFIDRPI FAWVIALVIM LAGGLSILSL PVNQYPAIAP PAIAVQVSYP GASAETVQDT VVQVIEQQMN GIDNLRYISS ESNSDGSMT ITVTFEQGTD PDIAQVQVQN KLQLATPLLP QEVQRQGIRV TKAVKNFLMV VGVVSTDGSM TKEDLSNYIV S NIQDPLSR TKGVGDFQVF GSQYSMRIWL DPAKLNSYQL TPGDVSSAIQ AQNVQISSGQ LGGLPAVKGQ QLNATIIGKT RL QTAEQFE NILLKVNPDG SQVRLKDVAD VGLGGQDYSI NAQFNGSPAS GIAIKLATGA NALDTAKAIR QTIANLEPFM PQG MKVVYP YDTTPVVSAS IHEVVKTLGE AILLVFLVMY LFLQNFRATL IPTIAVPVVL LGTFGVLAAF GFSINTLTMF GMVL AIGLL VDDAIVVVEN VERVMAEEGL SPREAARKSM GQIQGALVGI AMVLSAVFLP MAFFGGSTGV IYRQFSITIV SAMAL SVIV ALILTPALCA TMLKPIEKGD HGEHKGGFFG WFNRMFLSTT HGYERGVASI LKHRAPYLLI YVVIVAGMIW MFTRIP TAF LPDEDQGVLF AQVQTPPGSS AERTQVVVDS MREYLLEKES SSVSSVFTVT GFNFAGRGQS SGMAFIMLKP WEERPGG EN SVFELAKRAQ MHFFSFKDAM VFAFAPPSVL ELGNATGFDL FLQDQAGVGH EVLLQARNKF LMLAAQNPAL QRVRPNGM S DEPQYKLEID DEKASALGVS LADINSTVSI AWGSSYVNDF IDRGRVKRVY LQGRPDARMN PDDLSKWYVR NDKGEMVPF NAFATGKWEY GSPKLERYNG VPAMEILGEP APGLSSGDAM AAVEEIVKQL PKGVGYSWTG LSYEERLSGS QAPALYALSL LVVFLCLAA LYESWSIPFS VMLVVPLGVI GALLATSMRG LSNDVFFQVG LLTTIGLSAK NAILIVEFAK ELHEQGKGIV E AAIEACRM RLRPIVMTSL AFILGVVPLA ISTGAGSGSQ HAIGTGVIGG MVTATVLAIF WVPLFYVAVS TLFKDEASKQ QA SVEKGQL EHHHHHH

UniProtKB: Multidrug resistance protein MexB

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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

Concentration9.1 mg/mL
BufferpH: 7.5 / Component - Concentration: 50.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
VitrificationCryogen name: ETHANE / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 75000
Specialist opticsSpherical aberration corrector: CEOS Cs-corrector
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 95.0 K
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4080 pixel / Digitization - Dimensions - Height: 4080 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 8722 / Average exposure time: 2.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 535948
Startup modelType of model: OTHER
Details: Data from negative-stain EM single particle analysis
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 42338
FSC plot (resolution estimation)

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