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- EMDB-21228: Cryo-electron microscopy structures of a gonococcal multidrug eff... -

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Basic information

Entry
Database: EMDB / ID: EMD-21228
TitleCryo-electron microscopy structures of a gonococcal multidrug efflux pump illuminate a mechanism of drug recognition with ampicillin
Map dataStructure of a gonococcal multidrug efflux pump i
Sample
  • Complex: MtrD efflux pump
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: (2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-oxoethyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
KeywordsDrug / efflux / pump / MtrD / MEMBRANE PROTEIN / ampicillin
Function / homology
Function and homology information


xenobiotic transport / efflux transmembrane transporter activity / response to toxic substance / plasma membrane
Similarity search - Function
Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
: / Efflux pump membrane transporter
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsMoseng MA / Lyu M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI145069 United States
CitationJournal: mBio / Year: 2020
Title: Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance.
Authors: Meinan Lyu / Mitchell A Moseng / Jennifer L Reimche / Concerta L Holley / Vijaya Dhulipala / Chih-Chia Su / William M Shafer / Edward W Yu /
Abstract: is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in is the ultiple ... is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in is the ultiple ransferrable esistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene , encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.
History
DepositionJan 22, 2020-
Header (metadata) releaseFeb 12, 2020-
Map releaseJul 1, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vks
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21228.png

Downloads & links

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Map

FileDownload / File: emd_21228.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of a gonococcal multidrug efflux pump i
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 5
Minimum - Maximum-13.754369000000001 - 35.918823000000003
Average (Standard dev.)0.000000000001317 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z396.000396.000396.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-13.75435.9190.000

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Supplemental data

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Sample components

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Entire : MtrD efflux pump

EntireName: MtrD efflux pump
Components
  • Complex: MtrD efflux pump
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: (2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-oxoethyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid

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Supramolecule #1: MtrD efflux pump

SupramoleculeName: MtrD efflux pump / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)
Molecular weightTheoretical: 300 KDa

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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: Neisseria gonorrhoeae (bacteria)
Molecular weightTheoretical: 112.04243 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAKFFIDRPI FAWVISIFII AAGIFGIKSL PVSQYPSVAA PTITLHAIYP GASAQVMEGS VLSVIERNMN GVEGLDYMST SADSSGSGS VSLTFTPDTD ENLAQVEVQN KLSEVLSTLP ATVQQYGVTV SKARSNFLMI VMLSSDVQST EEMNDYAQRN V VPELQRIE ...String:
MAKFFIDRPI FAWVISIFII AAGIFGIKSL PVSQYPSVAA PTITLHAIYP GASAQVMEGS VLSVIERNMN GVEGLDYMST SADSSGSGS VSLTFTPDTD ENLAQVEVQN KLSEVLSTLP ATVQQYGVTV SKARSNFLMI VMLSSDVQST EEMNDYAQRN V VPELQRIE GVGQVRLFGA QRAMRIWVDP KKLQNYNLSF ADVGSALSAQ NIQISAGSIG SLPAVRGQTV TATVTAQGQL GT AEEFGNV ILRANTDGSN IYLKDVAKVG LGMEDYSSST RLNGVNTTGM AVMLSNSGNA MATAKAVKER LAVLEKYFPQ GMS WKTPYD TSKFVEISIE KVIHTLIEAM VLVFVVMYLF LQNIRYTLIP TIVVPISLLG GFAFISYMGM SINVLTMFAM ILVI GIVVD DAIVVVENVE RIMAGEGLPP KEATKKAMGQ ISGAVIGITA VLISVFVPLA MFSGAAGNIY KQFALTMASS IAFSA FLAL TLTPALCATM LKTIPKGHHE EKKGFFGWFN KKFDSWTHGY EGRVAKVLRK TFRMMVVYIG LAVVGVFLFM RLPTSF LPT EDQGFVMVSV QLPAGATKER TDATLAQVTQ LAKSIPEIEN IITVSGFSFS GSGQNMAMGF AILKDWNERT ASGSDAV AV AGKLTGMMMG TLKDGFGIAV VPPPILELGN GSGLSINLQD RNNTGHTALL AKRNELIQKM RASGLFDPST VRAGGLED S PQLKIDINRA AAAAQGVSFA DIRTALASAL SSSYVSDFPN QGRLQRVMVQ ADGDARMQPA DILNLTVPNS SGIAVPLSS IATVSWQMGT EQSVRFNGYP AMELSGSPAT GVSTGQAMEA VQKMVDELGS GYSLEWGGQS REEAKGGSQT IALYALAAVA VFLVLAALY ESWSIPLAVL LVMPLGLAGA AAGVTGRNLF EGLLGSVPSF ANDIYFQVGF VTVMGLSAKN AILIIEFAKD L QAQGKSAV EAALEAARLR FRPIIMTSFA FILGVVPLYI AGGASSASQR AIGTTVFWGM LIGTLLSVFL VPLFYVVVRK FF KETAHEH EM

UniProtKB: UNIPROTKB: A0A4T9VBR9

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Macromolecule #2: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 2 / Number of copies: 17 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #3: (2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-oxoethyl...

MacromoleculeName: (2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-oxoethyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
type: ligand / ID: 3 / Number of copies: 1 / Formula: AIX
Molecular weightTheoretical: 351.421 Da
Chemical component information

ChemComp-AIX:
(2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-oxoethyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid

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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

Concentration.7 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 10.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.5 K / Instrument: FEI VITROBOT MARK I / Details: blot 15 seconds before plunging.
DetailsMtrD embedded in nanodiscs

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ows


Details: AdeB multidrug efflux pump
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 81808

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6vks:
Cryo-electron microscopy structures of a gonococcal multidrug efflux pump illuminate a mechanism of drug recognition with ampicillin

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