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- EMDB-16659: Inward-facing conformation of the ABC transporter BmrA C436S/A582... -

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Basic information

Entry
Database: EMDB / ID: EMD-16659
TitleInward-facing conformation of the ABC transporter BmrA C436S/A582C cross-linked mutant
Map data
Sample
  • Complex: Homodimeric ATP-binding Cassette Transporter BmrA
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
KeywordsABC transporter / drug efflux pump / homodimer / membrane protein / antibiotic resistance
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: 3.14 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
DepositionFeb 7, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16659.png

Downloads & links

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Map

FileDownload / File: emd_16659.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 321. Å		1.07 Å/pix.
x 300 pix.
= 321. Å		1.07 Å/pix.
x 300 pix.
= 321. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.155
Minimum - Maximum-0.7911261 - 1.388648
Average (Standard dev.)0.00037480105 (±0.017963696)
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_16659_msk_1.map
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Half map: #1

Fileemd_16659_half_map_1.map
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Half map: #2

Fileemd_16659_half_map_2.map
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Sample components

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Entire : Homodimeric ATP-binding Cassette Transporter BmrA

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

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Supramolecule #1: Homodimeric ATP-binding Cassette Transporter BmrA

SupramoleculeName: Homodimeric ATP-binding Cassette Transporter BmrA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria)

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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: 66.340836 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 SYGLERDVTD AEIEKAAEMA YALNFIKELP NQFDTEVGER GIMLS GGQR QRIAIARALL RNPSILMLDE ATSSLDSQSE KSVQQALEVL MEGRTTIVIA HRLSTVVDAD QLLFVEKGEI TGRGTH HEL MASHGLYRDF AEQQLKMNCD 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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4S4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid
50.0 mMNaClSodium 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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 13937 / Average exposure time: 3.8 sec. / Average electron dose: 50.0 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: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 81000
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: 2057656
Startup modelType of model: OTHER / Details: Ab initio model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 343396
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 3D classificationNumber classes: 50 / Avg.num./class: 47623 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 90.1
Output model

PDB-8chb:
Inward-facing conformation of the ABC transporter BmrA C436S/A582C cross-linked mutant

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