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- EMDB-4460: Cryo-EM structure of Salmonella AcrB solubilised in the SMA copolymer -

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Basic information

Entry
Database: EMDB / ID: EMD-4460
TitleCryo-EM structure of Salmonella AcrB solubilised in the SMA copolymer
Map dataLocal resolution filtered map of Salmonella AcrB
Sample
  • Complex: Salmonella AcrB
    • Protein or peptide: Salmonella multidrug efflux transporter AcrB in a SMALP platform
Function / homology
Function and homology information


xenobiotic transport / efflux transmembrane transporter activity / membrane => GO:0016020 / plasma membrane
Similarity search - Function
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 / Efflux pump membrane transporter
Similarity search - Component
Biological speciesSalmonella (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsJohnson RM / Bavro VN / Postis V / Muench SP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust108372/A/15/Z United Kingdom
CitationJournal: Microorganisms / Year: 2020
Title: Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from .
Authors: Rachel M Johnson / Chiara Fais / Mayuriben Parmar / Harish Cheruvara / Robert L Marshall / Sophie J Hesketh / Matthew C Feasey / Paolo Ruggerone / Attilio V Vargiu / Vincent L G Postis / ...Authors: Rachel M Johnson / Chiara Fais / Mayuriben Parmar / Harish Cheruvara / Robert L Marshall / Sophie J Hesketh / Matthew C Feasey / Paolo Ruggerone / Attilio V Vargiu / Vincent L G Postis / Stephen P Muench / Vassiliy N Bavro /
Abstract: is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of ... is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the AcrB transporter remained unknown to-date, with much of our structural understanding coming from the orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of AcrB. Together, these reveal several important differences with respect to the protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.
History
DepositionDec 4, 2018-
Header (metadata) releaseApr 1, 2020-
Map releaseApr 1, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z12
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4460.png

Downloads & links

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Map

FileDownload / File: emd_4460.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution filtered map of Salmonella AcrB
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0196 / Movie #1: 0.04
Minimum - Maximum-0.096414484 - 0.15165184
Average (Standard dev.)0.002389971 (±0.009582522)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.00002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z214.000214.000214.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0960.1520.002

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Supplemental data

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Additional map: Post-processed (sharpened) cryo-EM map of Salmonella AcrB

Fileemd_4460_additional_1.map
AnnotationPost-processed (sharpened) cryo-EM map of Salmonella AcrB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo-EM map of Salmonella AcrB

Fileemd_4460_additional_2.map
AnnotationUnsharpened cryo-EM map of Salmonella AcrB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 for Salmonella AcrB

Fileemd_4460_half_map_1.map
AnnotationHalf map 1 for Salmonella AcrB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 for Salmonella AcrB

Fileemd_4460_half_map_2.map
AnnotationHalf map 2 for Salmonella AcrB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Salmonella AcrB

EntireName: Salmonella AcrB
Components
  • Complex: Salmonella AcrB
    • Protein or peptide: Salmonella multidrug efflux transporter AcrB in a SMALP platform

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Supramolecule #1: Salmonella AcrB

SupramoleculeName: Salmonella AcrB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Cryo-EM structure of Salmonella multidrug bacterial efflux pump AcrB solubilised in SMA co-polymer
Source (natural)Organism: Salmonella (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 340 KDa

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Macromolecule #1: Salmonella multidrug efflux transporter AcrB in a SMALP platform

MacromoleculeName: Salmonella multidrug efflux transporter AcrB in a SMALP platform
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPNFFIDRPI FAWVIAIIIM LAGGLAILKL PVAQYPTIAP PAVTISATYP GADAKTVQDT VTQVIEQNM NGIDNLMYMS SNSDSTGTVQ ITLTFESGTD ADIAQVQVQN KLQLAMPLLP Q EVQQQGVS VEKSSSSFLM VVGVINTDGT MTQEDISDYV AANMKDPISR ...String:
MPNFFIDRPI FAWVIAIIIM LAGGLAILKL PVAQYPTIAP PAVTISATYP GADAKTVQDT VTQVIEQNM NGIDNLMYMS SNSDSTGTVQ ITLTFESGTD ADIAQVQVQN KLQLAMPLLP Q EVQQQGVS VEKSSSSFLM VVGVINTDGT MTQEDISDYV AANMKDPISR TSGVGDVQLF GS QYAMRIW MNPTELTKYQ LTPVDVINAI KAQNAQVAAG QLGGTPPVKG QQLNASIIAQ TRL TSTDEF GKILLKVNQD GSQVRLRDVA KIELGGENYD VIAKFNGQPA SGLGIKLATG ANAL DTATA IRAELKKMEP FFPPGMKIVY PYDTTPFVKI SIHEVVKTLV EAIILVFLVM YLFLQ NFRA TLIPTIAVPV VLLGTFAVLA AFGFSINTLT MFGMVLAIGL LVDDAIVVVE NVERVM TEE GLPPKEATRK SMGQIQGALV GIAMVLSAVF IPMAFFGGST GAIYRQFSIT IVSAMAL SV LVALILTPAL CATMLKPVAK GDHGEGKKGF FGWFNRLFDK STHHYTDSVG NILRSTGR Y LLLYLIIVVG MAYLFVRLPS SFLPDEDQGV FLTMVQLPAG ATQERTQKVL DEVTDYYLN KEKANVESVF AVNGFGFAGR GQNTGIAFVS LKDWADRPGE KNKVEAITQR ATAAFSQIKD AMVFAFNLP AIVELGTATG FDFELIDQAG LGHEKLTQAR NQLFGEVAKY PDLLVGVRPN G LEDTPQFK IDIDQEKAQA LGVSISDINT TLGAAWGGSY VNDFIDRGRV KKVYVMSEAK YR MLPDDIN DWYVRGSDGQ MVPFSAFSSS RWEYGSPRLE RYNGLPSMEI LGQAAPGKST GEA MAMMEE LASKLPSGIG YDWTGMSYQE RLSGNQAPAL YAISLIVVFL CLAALYESWS IPFS VMLVV PLGVIGALLA ATFRGLTNDV YFQVGLLTTI GLSAKNAILI VEFAKDLMDK EGKGL VEAT LEAVRMRLRP ILMTSLAFML GVMPLVISSG AGSGAQNAVG TGVLGGMVTA TVLAIF FVP VFFVVVRRRF SRKSEDIEHS HSTEHR

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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

Concentration1.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium Chloride
50.0 mMC4H11NO3Tris

Details: Salmonella AcrB was prepared in buffer containing 50mM Tris (pH8) with 500 mM Nacl and 10% glycerol. For EM grids, the sample was diluted into buffer containing 50 mM Tris and 150 mM NaCl.
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were prepared using a Vitrobot Mark IV and blotted with Ash-free Whatman filter paper (No. 50) for 6 seconds using a blot force of 6..
DetailsSalmonella AcrB was prepared in buffer containing 50mM Tris (pH8) with 500 mM Nacl and 10% glycerol. For EM grids, the sample was diluted into buffer containing 50 mM Tris and 150 mM NaCl.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 3210 / Average exposure time: 10.0 sec. / Average electron dose: 61.7 e/Å2 / Details: 3,210 images were collected in a 72 hour period.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.0045 µm / Nominal defocus min: 0.0015 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 965863
Details: 1,410 particles were initially manually picked to generate 2D references which facilitated auto-picking.
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: EMDB MAP
EMDB ID:

3887


Details: The map was backfiltered to 60 A before being used in refinement/classification steps.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 105901
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 150 / Avg.num./class: 6500 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4zlj

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 305
Output model

PDB-6z12:
Salmonella AcrB solubilised in the SMA copolymer

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