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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 data
SampleMtrD efflux pump:
Efflux pump membrane transporter / (ligand) x 2
Function / homologyun:a0a4t9vbr9:
Function and homology information
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.
Validation ReportPDB-ID: 6vks

SummaryFull reportAbout validation report
History
DepositionJan 22, 2020-
Header (metadata) releaseFeb 12, 2020-
Map releaseJul 1, 2020-
UpdateJul 1, 2020-
Current statusJul 1, 2020Processing 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 360 pix.
= 396. Å
1.1 Å/pix.
x 360 pix.
= 396. Å
1.1 Å/pix.
x 360 pix.
= 396. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 3 / Movie #1: 5
Minimum - Maximum-13.754369 - 35.918823
Average (Standard dev.)0.00000000000 (±1)
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 / Number of components: 4

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Component #1: protein, MtrD efflux pump

ProteinName: MtrD efflux pump / Recombinant expression: No
MassExperimental: 300 kDa
SourceSpecies: Neisseria gonorrhoeae (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Efflux pump membrane transporter

ProteinName: Efflux pump membrane transporter / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 112.04243 kDa
SourceSpecies: Neisseria gonorrhoeae (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: ligand, PHOSPHATIDYLETHANOLAMINE

LigandName: PHOSPHATIDYLETHANOLAMINE / Number of Copies: 17 / Recombinant expression: No
MassTheoretical: 0.734039 kDa

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Component #4: ligand, (2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-...

LigandName: (2R,4S)-2-[(1R)-1-{[(2R)-2-amino-2-phenylacetyl]amino}-2-oxoethyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.351421 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.7 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 277.5 K / Humidity: 100 % / Details: blot 15 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 81808
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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