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- EMDB-40093: multi-drug efflux pump RE-CmeB bound with Ciprofloxacin -

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Basic information

Entry
Database: EMDB / ID: EMD-40093
Titlemulti-drug efflux pump RE-CmeB bound with Ciprofloxacin
Map datamulti-drug efflux pump RE-CmeB bound with Ciprofloxacin
Sample
  • Complex: RE-CmeB
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: 1-CYCLOPROPYL-6-FLUORO-4-OXO-7-PIPERAZIN-1-YL-1,4-DIHYDROQUINOLINE-3-CARBOXYLIC ACID
Keywordsefflux pump / membrane protein
Function / homology
Function and homology information


xenobiotic transport / efflux transmembrane transporter activity / 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
Biological speciesCampylobacter jejuni (Campylobacter)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 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: Microbiol Spectr / Year: 2023
Title: Cryo-Electron Microscopy Structures of a Campylobacter Multidrug Efflux Pump Reveal a Novel Mechanism of Drug Recognition and Resistance.
Authors: Zhemin Zhang / Nicholas Lizer / Zuowei Wu / Christopher E Morgan / Yuqi Yan / Qijing Zhang / Edward W Yu /
Abstract: Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and ...Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.
History
DepositionMar 16, 2023-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40093.png

Downloads & links

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Map

FileDownload / File: emd_40093.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmulti-drug efflux pump RE-CmeB bound with Ciprofloxacin
Projections & slices

Image control

Size
Brightness
Contrast
Others
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.30864498 - 0.7667275
Average (Standard dev.)0.00022276699 (±0.021234216)
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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Half map: Half Map 1

Fileemd_40093_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_40093_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RE-CmeB

EntireName: RE-CmeB
Components
  • Complex: RE-CmeB
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: 1-CYCLOPROPYL-6-FLUORO-4-OXO-7-PIPERAZIN-1-YL-1,4-DIHYDROQUINOLINE-3-CARBOXYLIC ACID

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Supramolecule #1: RE-CmeB

SupramoleculeName: RE-CmeB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Campylobacter jejuni (Campylobacter)

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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: Campylobacter jejuni (Campylobacter)
Molecular weightTheoretical: 114.122477 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFSKFFIERP IFASVVAIII SIAGIIGLAN LPVEQYPSLT PPTVQVSATY TGADAQTIAS TVATPIEDAI NGVDNMIYMD STSSPGQMK LTVYFNIGTD PDQAAIDVNN RISAATAKLP EAVKKLGVTV RKSSSTILEV VSVYSEDSSM NDIDIYNYVS L NILDELKR ...String:
MFSKFFIERP IFASVVAIII SIAGIIGLAN LPVEQYPSLT PPTVQVSATY TGADAQTIAS TVATPIEDAI NGVDNMIYMD STSSPGQMK LTVYFNIGTD PDQAAIDVNN RISAATAKLP EAVKKLGVTV RKSSSTILEV VSVYSEDSSM NDIDIYNYVS L NILDELKR IPGVGDASAI GNKNYSMRIW LEPDLLNKFG VTANDVINAV NDQNAQYATG KIGEEPVVNK SPQVISITMQ GR LQTPQEF ENIILRVNED KSFLRIKDVA KVEIGAEQYN STGRLNTSAA VPIIINLQSG ANAVNTAKLI NEKMQELSKN FPQ GLKYQI PYDTTIFVKA SIKEVIKTFV EALALVLVVM YLFLKNFKST IIPMIAVPVS LLGTFAVLYV LGFSINLLTL FALV LAIGI VVDDAIIVVE NIDRILHEDS NISVKDAAIK AMNEVSSPVI SIVLVLCAVF IPVSFISGFV GEIQRQFALT LAISV AISG FVALTLTPSL SALFLTRNES KPFYFIQKFN DFFDWSTSVF SSGVAYILKR TIRFVLVFCI MIGFIAYLFK IVPSSL VPS EDQGVIMSII NLPSGSSIHR TIEEVDTINK NATQMKEISS SVSLIGFDLF TSSLKENAAA VFFILKDWSQ REASSDQ II AQLFGQYAAD RNALSYFLNL PPIPGLSLTG GFEMYAQNKS GKDYDAIQQD VNKMLELART RKELANVRTT LDTSFPQY K LIIDRDKMKY YNLNMQDVFN TISATIGTYY VNDFPMLGKN FQVNIRALGD FRNTQDALKN IYIRSSDNKM IPLNSFLTL VRSAGPDDVK RFNLFPAALI QGDPAPGYTS GQAIDAIAEV AKQSLGDEYS IAWSGSAYQE VSSKGAGAYA FVLGMIFVFL ILAAQYERW LMPLAVITAV PFAVFGSILL VALRGFDNDI YFQTGLLLLI GLSAKNAILI IEFAMEERLK KGKSIFEAAI N AAKLRFRP IIMTSLAFTF GVLPMIFATG AGSASRHSLG TGLIGGMIAA STLAIFFVPL FFYLLENFNE WLDKKRGKVH E

UniProtKB: CmeB

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Macromolecule #2: 1-CYCLOPROPYL-6-FLUORO-4-OXO-7-PIPERAZIN-1-YL-1,4-DIHYDROQUINOLIN...

MacromoleculeName: 1-CYCLOPROPYL-6-FLUORO-4-OXO-7-PIPERAZIN-1-YL-1,4-DIHYDROQUINOLINE-3-CARBOXYLIC ACID
type: ligand / ID: 2 / Number of copies: 1 / Formula: CPF
Molecular weightTheoretical: 331.342 Da
Chemical component information

ChemComp-CPF:
1-CYCLOPROPYL-6-FLUORO-4-OXO-7-PIPERAZIN-1-YL-1,4-DIHYDROQUINOLINE-3-CARBOXYLIC ACID / antibiotic*YM

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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: 37.9 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 43849
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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