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- EMDB-10185: Cryo-EM structure of Escherichia coli AcrB and DARPin in Saposin ... -

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Basic information

Entry
Database: EMDB / ID: EMD-10185
TitleCryo-EM structure of Escherichia coli AcrB and DARPin in Saposin A-nanodisc
Map data
Sample
  • Complex: Ternary complex of AcrB and DARPin in SaposinA-nanodisc
    • Complex: Multidrug efflux pump subunit AcrB
      • Protein or peptide: Multidrug efflux pump subunit AcrB
    • Complex: DARPin
      • Protein or peptide: DARPin
KeywordsRND transporter / efflux pump / drug transport / antibiotic resistance / lipid nanodisc / MEMBRANE PROTEIN
Function / homology
Function and homology information


alkane transmembrane transporter activity / alkane transport / xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / enterobactin transport / enterobactin transmembrane transporter activity / periplasmic side of plasma membrane / bile acid transmembrane transporter activity / bile acid and bile salt transport / xenobiotic transport ...alkane transmembrane transporter activity / alkane transport / xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / enterobactin transport / enterobactin transmembrane transporter activity / periplasmic side of plasma membrane / bile acid transmembrane transporter activity / bile acid and bile salt transport / xenobiotic transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / fatty acid transport / response to toxic substance / response to xenobiotic stimulus / response to antibiotic / identical protein binding / membrane / 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
Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / synthetic construct (others) / Escherichia coli K12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsSzewczak-Harris A / Du D
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
European Research Council742210 United Kingdom
CitationJournal: Structure / Year: 2020
Title: Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment.
Authors: Dijun Du / Arthur Neuberger / Mona Wu Orr / Catherine E Newman / Pin-Chia Hsu / Firdaus Samsudin / Andrzej Szewczak-Harris / Leana M Ramos / Mekdes Debela / Syma Khalid / Gisela Storz / Ben F Luisi /
Abstract: The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic ...The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.
History
DepositionAug 5, 2019-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sgu
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10185.png

Downloads & links

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Map

FileDownload / File: emd_10185.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.11146742 - 0.19031174
Average (Standard dev.)-0.000015067448 (±0.0057090856)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z318.000318.000318.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1110.190-0.000

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Supplemental data

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Mask #1

Fileemd_10185_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10185_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10185_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of AcrB and DARPin in SaposinA-nanodisc

EntireName: Ternary complex of AcrB and DARPin in SaposinA-nanodisc
Components
  • Complex: Ternary complex of AcrB and DARPin in SaposinA-nanodisc
    • Complex: Multidrug efflux pump subunit AcrB
      • Protein or peptide: Multidrug efflux pump subunit AcrB
    • Complex: DARPin
      • Protein or peptide: DARPin

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Supramolecule #1: Ternary complex of AcrB and DARPin in SaposinA-nanodisc

SupramoleculeName: Ternary complex of AcrB and DARPin in SaposinA-nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Multidrug efflux pump subunit AcrB

SupramoleculeName: Multidrug efflux pump subunit AcrB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: DARPin

SupramoleculeName: DARPin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Multidrug efflux pump subunit AcrB

MacromoleculeName: Multidrug efflux pump subunit AcrB / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K12 (bacteria)
Molecular weightTheoretical: 113.66518 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString: MPNFFIDRPI FAWVIAIIIM LAGGLAILKL PVAQYPTIAP PAVTISASYP GADAKTVQDT VTQVIEQNMN GIDNLMYMSS NSDSTGTVQ ITLTFESGTD ADIAQVQVQN KLQLAMPLLP QEVQQQGVSV EKSSSSFLMV VGVINTDGTM TQEDISDYVA A NMKDAISR ...String:
MPNFFIDRPI FAWVIAIIIM LAGGLAILKL PVAQYPTIAP PAVTISASYP GADAKTVQDT VTQVIEQNMN GIDNLMYMSS NSDSTGTVQ ITLTFESGTD ADIAQVQVQN KLQLAMPLLP QEVQQQGVSV EKSSSSFLMV VGVINTDGTM TQEDISDYVA A NMKDAISR TSGVGDVQLF GSQYAMRIWM NPNELNKFQL TPVDVITAIK AQNAQVAAGQ LGGTPPVKGQ QLNASIIAQT RL TSTEEFG KILLKVNQDG SRVLLRDVAK IELGGENYDI IAEFNGQPAS GLGIKLATGA NALDTAAAIR AELAKMEPFF PSG LKIVYP YDTTPFVKIS IHEVVKTLVE AIILVFLVMY LFLQNFRATL IPTIAVPVVL LGTFAVLAAF GFSINTLTMF GMVL AIGLL VDDAIVVVEN VERVMAEEGL PPKEATRKSM GQIQGALVGI AMVLSAVFVP MAFFGGSTGA IYRQFSITIV SAMAL SVLV ALILTPALCA TMLKPIAKGD HGEGKKGFFG WFNRMFEKST HHYTDSVGGI LRSTGRYLVL YLIIVVGMAY LFVRLP SSF LPDEDQGVFM TMVQLPAGAT QERTQKVLNE VTHYYLTKEK NNVESVFAVN GFGFAGRGQN TGIAFVSLKD WADRPGE EN KVEAITMRAT RAFSQIKDAM VFAFNLPAIV ELGTATGFDF ELIDQAGLGH EKLTQARNQL LAEAAKHPDM LTSVRPNG L EDTPQFKIDI DQEKAQALGV SINDINTTLG AAWGGSYVND FIDRGRVKKV YVMSEAKYRM LPDDIGDWYV RAADGQMVP FSAFSSSRWE YGSPRLERYN GLPSMEILGQ AAPGKSTGEA MELMEQLASK LPTGVGYDWT GMSYQERLSG NQAPSLYAIS LIVVFLCLA ALYESWSIPF SVMLVVPLGV IGALLAATFR GLTNDVYFQV GLLTTIGLSA KNAILIVEFA KDLMDKEGKG L IEATLDAV RMRLRPILMT SLAFILGVMP LVISTGAGSG AQNAVGTGVM GGMVTATVLA IFFVPVFFVV VRRRFSRKNE DI EHSHTVD HH

UniProtKB: Multidrug efflux pump subunit AcrB

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Macromolecule #2: DARPin

MacromoleculeName: DARPin / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.317566 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString:
MRGSHHHHHH GSDLGKKLLE AARAGRDDEV RILMANGADV NAADVVGWTP LHLAAYWGHL EIVEVLLKNG ADVNAYDTLG STPLHLAAH FGHLEIVEVL LKNGADVNAK DDNGITPLHL AANRGHLEIV EVLLKYGADV NAQDKFGKTA FDISINNGNE D LAEILQKL N

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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

Concentration2.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Tris
150.0 mMNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2156 / Average exposure time: 20.0 sec. / Average electron dose: 1.335 e/Å2
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: 3.4 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 75000
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

Startup modelType of model: PDB ENTRY
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 414786
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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