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- EMDB-28857: Gentamicin bound aminoglycoside efflux pump AcrD -

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Basic information

Entry
Database: EMDB / ID: EMD-28857
TitleGentamicin bound aminoglycoside efflux pump AcrD
Map data
Sample
  • Complex: AcrD
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: (2R,3R,4R,5R)-2-((1S,2S,3R,4S,6R)-4,6-DIAMINO-3-((2R,3R,6S)-3-AMINO-6-(AMINOMETHYL)-TETRAHYDRO-2H-PYRAN-2-YLOXY)-2-HYDR OXYCYCLOHEXYLOXY)-5-METHYL-4-(METHYLAMINO)-TETRAHYDRO-2H-PYRAN-3,5-DIOL
Keywordsaminoglycoside efflux pump / AcrD / E.coli / MEMBRANE PROTEIN
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 speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsZhang Z
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI145069 United States
CitationJournal: mBio / Year: 2023
Title: Cryo-EM Structures of AcrD Illuminate a Mechanism for Capturing Aminoglycosides from Its Central Cavity.
Authors: Zhemin Zhang / Christopher E Morgan / Meng Cui / Edward W Yu /
Abstract: The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance ...The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance to aminoglycoside-based drugs by actively extruding these noxious compounds out of E. coli cells. AcrD can also mediate resistance to a limited range of other amphiphilic agents, including bile acids, novobiocin, and fusidic acids. As there is no structural information available for any aminoglycoside-specific RND pump, here we describe cryo-electron microscopy (cryo-EM) structures of AcrD in the absence and presence of bound gentamicin. These structures provide new information about the RND superfamily of efflux pumps, specifically, that three negatively charged residues central to the aminoglycoside-binding site are located within the ceiling of the central cavity of the AcrD trimer. Thus, it is likely that AcrD is capable of picking up aminoglycosides via this central cavity. Through the combination of cryo-EM structural determination, mutagenesis analysis, and molecular simulation, we show that charged residues are critically important for this pump to shuttle drugs directly from the central cavity to the funnel of the AcrD trimer for extrusion. Here, we report cryo-EM structures of the AcrD aminoglycoside efflux pump in the absence and presence of bound gentamicin, posing the possibility that this pump is capable of capturing aminoglycosides from the central cavity of the AcrD trimer. The results indicate that AcrD utilizes charged residues to bind and export drugs, mediating resistance to these antibiotics.
History
DepositionNov 11, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28857.png

Downloads & links

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Map

FileDownload / File: emd_28857.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 352 pix.
= 376.64 Å		1.07 Å/pix.
x 352 pix.
= 376.64 Å		1.07 Å/pix.
x 352 pix.
= 376.64 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.6467138 - 1.4782466
Average (Standard dev.)0.0000091610445 (±0.047488037)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 376.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_28857_additional_1.map
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Half map: #2

Fileemd_28857_half_map_1.map
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Half map: #1

Fileemd_28857_half_map_2.map
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Sample components

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Entire : AcrD

EntireName: AcrD
Components
  • Complex: AcrD
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: (2R,3R,4R,5R)-2-((1S,2S,3R,4S,6R)-4,6-DIAMINO-3-((2R,3R,6S)-3-AMINO-6-(AMINOMETHYL)-TETRAHYDRO-2H-PYRAN-2-YLOXY)-2-HYDR OXYCYCLOHEXYLOXY)-5-METHYL-4-(METHYLAMINO)-TETRAHYDRO-2H-PYRAN-3,5-DIOL

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Supramolecule #1: AcrD

SupramoleculeName: AcrD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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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: Escherichia coli (E. coli)
Molecular weightTheoretical: 113.139891 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MANFFIDRPI FAWVLAILLC LTGTLAIFSL PVEQYPDLAP PNVRVTANYP GASAQTLENT VTQVIEQNMT GLDNLMYMSS QSSGTGQAS VTLSFKAGTD PDEAVQQVQN QLQSAMRKLP QAVQNQGVTV RKTGDTNILT IAFVSTDGSM DKQDIADYVA S NIQDPLSR ...String:
MANFFIDRPI FAWVLAILLC LTGTLAIFSL PVEQYPDLAP PNVRVTANYP GASAQTLENT VTQVIEQNMT GLDNLMYMSS QSSGTGQAS VTLSFKAGTD PDEAVQQVQN QLQSAMRKLP QAVQNQGVTV RKTGDTNILT IAFVSTDGSM DKQDIADYVA S NIQDPLSR VNGVGDIDAY GSQYSMRIWL DPAKLNSFQM TAKDVTDAIE SQNAQIAVGQ LGGTPSVDKQ ALNATINAQS LL QTPEQFR DITLRVNQDG SEVRLGDVAT VEMGAEKYDY LSRFNGKPAS GLGVKLASGA NEMATAELVL NRLDELAQYF PHG LEYKVA YETTSFVKAS IEDVVKTLLE AIALVFLVMY LFLQNFRATL IPTIAVPVVL MGTFSVLYAF GYSVNTLTMF AMVL AIGLL VDDAIVVVEN VERIMSEEGL TPREATRKSM GQIQGALVGI AMVLSAVFVP MAFFGGTTGA IYRQFSITIV AAMVL SVLV AMILTPALCA TLLKPLKKGE HHGQKGFFAW FNQMFNRNAE RYEKGVAKIL HRSLRWIVIY VLLLGGMVFL FLRLPT SFL PLEDRGMFTT SVQLPSGSTQ QQTLKVVEQI EKYYFTHEKD NIMSVFATVG SGPGGNGQNV ARMFIRLKDW SERDSKT GT SFAIIERATK AFNQIKEARV IASSPPAISG LGSSAGFDME LQDHAGAGHD ALMAARNQLL ALAAENPELT RVRHNGLD D SPQLQIDIDQ RKAQALGVAI DDINDTLQTA WGSSYVNDFM DRGRVKKVYV QAAAPYRMLP DDINLWYVRN KDGGMVPFS AFATSRWETG SPRLERYNGY SAVEIVGEAA PGVSTGTAMD IMESLVKQLP NGFGLEWTAM SYQERLSGAQ APALYAISLL VVFLCLAAL YESWSVPFSV MLVVPLGVIG ALLATWMRGL ENDVYFQVGL LTVIGLSAKN AILIVEFANE MNQKGHDLFE A TLHACRQR LRPILMTSLA FIFGVLPMAT STGAGSGGQH AVGTGVMGGM ISATILAIYF VPLFFVLVRR RFPLKPRPE

UniProtKB: Efflux pump membrane transporter

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Macromolecule #2: (2R,3R,4R,5R)-2-((1S,2S,3R,4S,6R)-4,6-DIAMINO-3-((2R,3R,6S)-3-AMI...

MacromoleculeName: (2R,3R,4R,5R)-2-((1S,2S,3R,4S,6R)-4,6-DIAMINO-3-((2R,3R,6S)-3-AMINO-6-(AMINOMETHYL)-TETRAHYDRO-2H-PYRAN-2-YLOXY)-2-HYDR OXYCYCLOHEXYLOXY)-5-METHYL-4-(METHYLAMINO)-TETRAHYDRO-2H-PYRAN-3,5-DIOL
type: ligand / ID: 2 / Number of copies: 1 / Formula: LLL
Molecular weightTheoretical: 449.542 Da

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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: 35.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 36919
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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