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- EMDB-24732: Multidrug Efflux pump AdeJ with TP-6076 bound -

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Basic information

Entry
Database: EMDB / ID: EMD-24732
TitleMultidrug Efflux pump AdeJ with TP-6076 bound
Map data
Sample
  • Complex: multidrug efflux pump AdeJ
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: (4S,4aS,5aR,12aS)-4-(diethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-[(2S)-pyrrolidin-2-yl]-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
Keywordsmultidrug efflux pump / MEMBRANE PROTEIN-ANTIBIOTIC complex
Function / homologyHydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / plasma membrane / Efflux pump membrane transporter
Function and homology information
Biological speciesAcinetobacter baumannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 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: An Analysis of the Novel Fluorocycline TP-6076 Bound to Both the Ribosome and Multidrug Efflux Pump AdeJ from Acinetobacter baumannii.
Authors: Christopher E Morgan / Zhemin Zhang / Robert A Bonomo / Edward W Yu /
Abstract: Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a ...Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a number of resistance determinants to evade current antibiotics. One of the major resistance mechanisms employed by these pathogens is the use of multidrug efflux pumps. These pumps extrude xenobiotics directly out of bacterial cells, resulting in treatment failures when common antibiotics are administered. Here, the structure of the novel tetracycline antibiotic TP-6076, bound to both the cinetobacter rug fflux pump AdeJ and the ribosome from Acinetobacter baumannii, using single-particle cryo-electron microscopy (cryo-EM), is elucidated. In this work, the structure of the AdeJ-TP-6076 complex is solved, and we show that AdeJ utilizes a network of hydrophobic interactions to recognize this fluorocycline. Concomitant with this, we elucidate three structures of TP-6076 bound to the A. baumannii ribosome and determine that its binding is stabilized largely by electrostatic interactions. We then compare the differences in binding modes between TP-6076 and the related tetracycline antibiotic eravacycline in both targets. These differences suggest that modifications to the tetracycline core may be able to alter AdeJ binding while maintaining interactions with the ribosome. Together, this work highlights how different mechanisms are used to stabilize the binding of tetracycline-based compounds to unique bacterial targets and provides guidance for the future clinical development of tetracycline antibiotics. Treatment of antibiotic-resistant organisms such as A. baumannii represents an ongoing issue for modern medicine. The multidrug efflux pump AdeJ serves as a major resistance determinant in A. baumannii through its action of extruding antibiotics from the cell. In this work, we use cryo-EM to show how AdeJ recognizes the experimental tetracycline antibiotic TP-6076 and prevents this drug from interacting with the A. baumannii ribosome. Since AdeJ and the ribosome use different binding modes to stabilize interactions with TP-6076, exploiting these differences may guide future drug development for combating antibiotic-resistant A. baumannii and potentially other strains of MDR bacteria.
History
DepositionAug 24, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ry3
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24732.png

Downloads & links

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Map

FileDownload / File: emd_24732.map.gz / Format: CCP4 / Size: 8.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 123 pix.
= 132.84 Å		1.08 Å/pix.
x 123 pix.
= 132.84 Å		1.08 Å/pix.
x 149 pix.
= 160.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11 / Movie #1: 0.2
Minimum - Maximum-1.7248108 - 3.0999274
Average (Standard dev.)-0.000000000002473 (±0.21724999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin110112116
Dimensions123149123
Spacing123123149
CellA: 132.84001 Å / B: 132.84001 Å / C: 160.92001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z123123149
origin x/y/z0.0000.0000.000
length x/y/z132.840132.840160.920
α/β/γ90.00090.00090.000
start NX/NY/NZ116110112
NX/NY/NZ123123149
MAP C/R/S321
start NC/NR/NS112110116
NC/NR/NS149123123
D min/max/mean-1.7253.100-0.000

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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: (4S,4aS,5aR,12aS)-4-(diethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-[(2S)-pyrrolidin-2-yl]-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

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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)

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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 (E. coli)
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

UniProtKB: Efflux pump membrane transporter

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Macromolecule #2: (4S,4aS,5aR,12aS)-4-(diethylamino)-3,10,12,12a-tetrahydroxy-1,11-...

MacromoleculeName: (4S,4aS,5aR,12aS)-4-(diethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-[(2S)-pyrrolidin-2-yl]-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
type: ligand / ID: 2 / Number of copies: 1 / Formula: 80P
Molecular weightTheoretical: 579.565 Da
Chemical component information

ChemComp-80P:
(4S,4aS,5aR,12aS)-4-(diethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-[(2S)-pyrrolidin-2-yl]-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 36.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 68346
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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