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- EMDB-18535: Inward-facing conformation of the ABC transporter BmrA -

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Basic information

Entry
Database: EMDB / ID: EMD-18535
TitleInward-facing conformation of the ABC transporter BmrA
Map data
Sample
  • Complex: ABC transporter BmrA
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
KeywordsABC transporter / drug efflux pump / homodimer / membrane protein / antibiotic resistance / TRANSPORT PROTEIN
Function / homology
Function and homology information


ATPase-coupled lipid transmembrane transporter activity / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ABC-type transporter activity / transmembrane transport / response to antibiotic / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. ...Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsHanssen E / Valimehr S / Di Cesare M / Kaplan E / Orelle C / Jault JM
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0023 France
CitationJournal: J Biol Chem / Year: 2024
Title: The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.
Authors: Margot Di Cesare / Elise Kaplan / Julia Rendon / Guillaume Gerbaud / Sepideh Valimehr / Alexia Gobet / Thu-Anh Thi Ngo / Vincent Chaptal / Pierre Falson / Marlène Martinho / Pierre Dorlet / ...Authors: Margot Di Cesare / Elise Kaplan / Julia Rendon / Guillaume Gerbaud / Sepideh Valimehr / Alexia Gobet / Thu-Anh Thi Ngo / Vincent Chaptal / Pierre Falson / Marlène Martinho / Pierre Dorlet / Eric Hanssen / Jean-Michel Jault / Cédric Orelle /
Abstract: ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug ...ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.
History
DepositionSep 28, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18535.png

Downloads & links

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Map

FileDownload / File: emd_18535.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.46044722 - 0.8905742
Average (Standard dev.)0.00044096773 (±0.016642656)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18535_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18535_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18535_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : ABC transporter BmrA

EntireName: ABC transporter BmrA
Components
  • Complex: ABC transporter BmrA
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA

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Supramolecule #1: ABC transporter BmrA

SupramoleculeName: ABC transporter BmrA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: bmrA, yvcC, BSU34820

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Macromolecule #1: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA

MacromoleculeName: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 66.324836 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPTKKQKSKS KLKPFFALVR RTNPSYGKLA FALALSVVTT LVSLLIPLLT KQLVDGFSMS NLSGTQIGLI ALVFFVQAGL SAYATYALN YNGQKIISGL RELLWKKLIK LPVSYFDTNA SGETVSRVTN DTMVVKELIT THISGFITGI ISVIGSLTIL F IMNWKLTL ...String:
MPTKKQKSKS KLKPFFALVR RTNPSYGKLA FALALSVVTT LVSLLIPLLT KQLVDGFSMS NLSGTQIGLI ALVFFVQAGL SAYATYALN YNGQKIISGL RELLWKKLIK LPVSYFDTNA SGETVSRVTN DTMVVKELIT THISGFITGI ISVIGSLTIL F IMNWKLTL LVLVVVPLAA LILVPIGRKM FSISRETQDE TARFTGLLNQ ILPEIRLVKA SNAEDVEYGR GKMGISSLFK LG VREAKVQ SLVGPLISLV LMAALVAVIG YGGMQVSSGE LTAGALVAFI LYLFQIIMPM GQITTFFTQL QKSIGATERM IEI LAEEEE DTVTGKQIEN AHLPIQLDRV SFGYKPDQLI LKEVSAVIEA GKVTAIVGPS GGGKTTLFKL LERFYSPTAG TIRL GDEPV DTYSLESWRE HIGYVSQESP LMSGTIRENI CYGLERDVTD AEIEKAAEMA YALNFIKELP NQFDTEVGER GIMLS GGQR QRIAIARALL RNPSILMLDE ATSSLDSQSE KSVQQALEVL MEGRTTIVIA HRLSTVVDAD QLLFVEKGEI TGRGTH HEL MASHGLYRDF AEQQLKMNAD LENKAGVDKL AAALEHHHHH H

UniProtKB: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA

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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: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4S4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid
50.0 mMNaClSodium chlorideSodium Chloride
0.035 %C24H46O11n-Dodecyl-B-D-maltopyranoside
0.03 %C24H39NaO5Sodium Cholate

Details: pH adjustment with sodium hydroxide
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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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: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3972311
Startup modelType of model: OTHER / Details: Ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF
Software: (Name: cryoSPARC (ver. 3.3.1), cryoSPARC (ver. 4.1.1))
Number images used: 1043614
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8chb


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting was done using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8qoe:
Inward-facing conformation of the ABC transporter BmrA

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