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- EMDB-23663: Multidrug Efflux pump AdeJ with Eravacycline bound -

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Basic information

Entry
Database: EMDB / ID: EMD-23663
TitleMultidrug Efflux pump AdeJ with Eravacycline bound
Map dataMultidrug Efflux pump AdeJ with Eravacylcine bound
Sample
  • Complex: multidrug efflux pump AdeJ
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
  • Ligand: Eravacycline
Function / homologyHydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / xenobiotic transport / efflux transmembrane transporter activity / plasma membrane / Efflux pump membrane transporter
Function and homology information
Biological speciesAcinetobacter baumannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsZhang Z
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: mBio / Year: 2021
Title: Cryo-EM Determination of Eravacycline-Bound Structures of the Ribosome and the Multidrug Efflux Pump AdeJ of Acinetobacter baumannii.
Authors: Zhemin Zhang / Christopher E Morgan / Robert A Bonomo / Edward W Yu /
Abstract: Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections ...Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the cinetobacter rug fflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.
History
DepositionMar 22, 2021-
Header (metadata) releaseMay 19, 2021-
Map releaseMay 19, 2021-
UpdateMar 2, 2022-
Current statusMar 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.216
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.216
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7m4p
  • Surface level: 0.216
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23663.png

Downloads & links

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Map

FileDownload / File: emd_23663.map.gz / Format: CCP4 / Size: 10.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMultidrug Efflux pump AdeJ with Eravacylcine bound
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.216 / Movie #1: 0.216
Minimum - Maximum-1.8649024 - 2.9686458
Average (Standard dev.)-4.0158247e-13 (±0.20173334)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin106106112
Dimensions130160133
Spacing133130160
CellA: 143.64 Å / B: 140.40001 Å / C: 172.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z133130160
origin x/y/z0.0000.0000.000
length x/y/z143.640140.400172.800
α/β/γ90.00090.00090.000
start NX/NY/NZ112106106
NX/NY/NZ133130160
MAP C/R/S321
start NC/NR/NS106106112
NC/NR/NS160130133
D min/max/mean-1.8652.969-0.000

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Supplemental data

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Sample components

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Entire : multidrug efflux pump AdeJ

EntireName: multidrug efflux pump AdeJ
Components
  • Complex: multidrug efflux pump AdeJ
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
  • Ligand: Eravacycline

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Supramolecule #1: multidrug efflux pump AdeJ

SupramoleculeName: multidrug efflux pump AdeJ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Acinetobacter baumannii (bacteria)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Efflux pump membrane transporter

MacromoleculeName: Efflux pump membrane transporter / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Acinetobacter baumannii (bacteria)
Molecular weightTheoretical: 114.637781 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MAQFFIHRPI FAWVIALVIM LAGILTLTKM PIAQYPTIAP PTVTIAATYP GASAETVENT VTQIIEQQMN GLDGLRYISS NSAGNGQAS IQLNFEQGVD PDIAQVQVQN KLQSATALLP EDVQRQGVTV TKSGASFLQV IAFYSPDNNL SDSDIKDYVN S SIKEPLSR ...String:
MAQFFIHRPI FAWVIALVIM LAGILTLTKM PIAQYPTIAP PTVTIAATYP GASAETVENT VTQIIEQQMN GLDGLRYISS NSAGNGQAS IQLNFEQGVD PDIAQVQVQN KLQSATALLP EDVQRQGVTV TKSGASFLQV IAFYSPDNNL SDSDIKDYVN S SIKEPLSR VAGVGEVQVF GGSYAMRIWL DPAKLTSYQL TPSDIATALQ AQNSQVAVGQ LGGAPAVQGQ VLNATVNAQS LL QTPEQFK NIFLKNTASG AEVRLKDVAR VELGSDNYQF DSKFNGKPAA GLAIKIATGA NALDTAEAVE QRLSELRKNY PTG LADKLA YDTTPFIRLS IESVVHTLIE AVILVFIVMF LFLQNWRATI IPTLAVPVVV LGTFAVINIF GFSINTLTMF AMVL AIGLL VDDAIVVVEN VERVMSEDHT DPVTATSRSM QQISGALVGI TSVLTAVFVP MAFFGGTTGV IYRQFSITLV TAMVL SLIV ALTFTPALCA TILKQHDPNK EPSNNIFARF FRSFNNGFDR MSHSYQNGVS RMLKGKIFSG VLYAVVVALL VFLFQK LPS SFLPEEDQGV VMTLVQLPPN ATLDRTGKVI DTMTNFFMNE KDTVESIFTV SGFSFTGVGQ NAGIGFVKLK DWSKRTT PE TQIGSLIQRG MALNMIIKDA SYVMPLQLPA MPELGVTAGF NLQLKDSSGQ GHEKLIAARN TILGLASQDK RLVGVRPN G QEDTPQYQIN VDQAQAGAMG VSIAEINNTM RIAWGGSYIN DFVDRGRVKK VYVQGDAGSR MMPEDLNKWY VRNNKGEMV PFSAFATGEW TYGSPRLERY NGVSSVNIQG TPAPGVSSGD AMKAMEEIIG KLPSMGLQGF DYEWTGLSLE ERESGAQAPF LYALSLLIV FLCLAALYES WSIPFSVLLV VPLGVIGAIV LTYLGMIIKG DPNLSNNIYF QVAIIAVIGL SAKNAILIVE F AKELQEKG EDLLDATLHA AKMRLRPIIM TTLAFGFGVL PLALSTGAGA GSQHSVGFGV LGGVLSATFL GIFFIPVFYV WI RSIFKYK PKTINTQEHK S

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Macromolecule #2: 1,2-Distearoyl-sn-glycerophosphoethanolamine

MacromoleculeName: 1,2-Distearoyl-sn-glycerophosphoethanolamine / type: ligand / ID: 2 / Number of copies: 8 / Formula: 3PE
Molecular weightTheoretical: 748.065 Da
Chemical component information

ChemComp-3PE:
1,2-Distearoyl-sn-glycerophosphoethanolamine / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #3: Eravacycline

MacromoleculeName: Eravacycline / type: ligand / ID: 3 / Number of copies: 1 / Formula: YQM
Molecular weightTheoretical: 558.555 Da
Chemical component information

ChemComp-YQM:
Eravacycline / antibiotic*YM / Eravacycline

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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: 7.5
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 K3 BIOQUANTUM (6k x 4k) / Average electron dose: 36.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 95451

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