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- EMDB-10183: Cryo-EM structure of Escherichia coli AcrBZ and DARPin in Saposin... -

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Basic information

Entry
Database: EMDB / ID: EMD-10183
TitleCryo-EM structure of Escherichia coli AcrBZ and DARPin in Saposin A-nanodisc
Map data
Sample
  • Complex: Ternary complex of AcrBZ and DARPin in Saposin A-nanodisc
    • Complex: Multidrug efflux pump subunit AcrB
      • Protein or peptide: Multidrug efflux pump subunit AcrB
    • Complex: DARPin
      • Protein or peptide: DARPin
    • Complex: Multidrug efflux pump accessory protein AcrZ
      • Protein or peptide: Multidrug efflux pump accessory protein AcrZ
Function / homology
Function and homology information


xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / periplasmic side of plasma membrane / xenobiotic detoxification by transmembrane export across the plasma membrane / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / outer membrane-bounded periplasmic space / membrane => GO:0016020 / response to antibiotic / membrane ...xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / periplasmic side of plasma membrane / xenobiotic detoxification by transmembrane export across the plasma membrane / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / outer membrane-bounded periplasmic space / membrane => GO:0016020 / response to antibiotic / membrane / identical protein binding / plasma membrane
Similarity search - Function
Multidrug efflux pump accessory protein AcrZ / Multidrug efflux pump-associated protein AcrZ / 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 accessory protein AcrZ / Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / synthetic construct (others) / Shigella boydii 965-58 (bacteria) / Escherichia coli K12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSzewczak-Harris A / Du D / Newman C / Neuberger A / Luisi BF
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-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sgs
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10183.png

Downloads & links

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Map

FileDownload / File: emd_10183.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.17104171 - 0.25545758
Average (Standard dev.)-0.00000120111 (±0.009158099)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 327.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z327.000327.000327.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1710.255-0.000

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Supplemental data

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Mask #1

Fileemd_10183_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10183_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10183_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of AcrBZ and DARPin in Saposin A-nanodisc

EntireName: Ternary complex of AcrBZ and DARPin in Saposin A-nanodisc
Components
  • Complex: Ternary complex of AcrBZ and DARPin in Saposin A-nanodisc
    • Complex: Multidrug efflux pump subunit AcrB
      • Protein or peptide: Multidrug efflux pump subunit AcrB
    • Complex: DARPin
      • Protein or peptide: DARPin
    • Complex: Multidrug efflux pump accessory protein AcrZ
      • Protein or peptide: Multidrug efflux pump accessory protein AcrZ

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Supramolecule #1: Ternary complex of AcrBZ and DARPin in Saposin A-nanodisc

SupramoleculeName: Ternary complex of AcrBZ and DARPin in Saposin A-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)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (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)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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Supramolecule #4: Multidrug efflux pump accessory protein AcrZ

SupramoleculeName: Multidrug efflux pump accessory protein AcrZ / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Shigella boydii 965-58 (bacteria)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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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

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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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Macromolecule #3: Multidrug efflux pump accessory protein AcrZ

MacromoleculeName: Multidrug efflux pump accessory protein AcrZ / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Shigella boydii 965-58 (bacteria)
Molecular weightTheoretical: 5.304423 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString:
MLELLKSLVF AVIMVPVVMA IILGLIYGLG EVFNIFSGVG KKDQPGQNH

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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 chlorideSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1356 / Average exposure time: 60.0 sec. / Average electron dose: 26.25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.08)
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 94507
FSC plot (resolution estimation)

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