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- EMDB-51550: BmrA E504A in complex with Hoechst33342 -

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Basic information

Entry
Database: EMDB / ID: EMD-51550
TitleBmrA E504A in complex with Hoechst33342
Map datasharpened map by cryosparc v4.5
Sample
  • Complex: BmrA E504A in complex with Hoechst33342
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
  • Ligand: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE
Keywordstransporter complex with Hoechst33342 / MEMBRANE 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: 3.6 Å
AuthorsMoissonnier L / Zarkadas E / Schoehn G / Falson P / Chaptal V
Funding support France, 2 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0023-01 France
Agence Nationale de la Recherche (ANR)ANR-23-CE11-0031 France
CitationJournal: Nat Commun / Year: 2025
Title: Rhodamine6G and Hœchst33342 narrow BmrA conformational spectrum for a more efficient use of ATP.
Authors: A Gobet / L Moissonnier / E Zarkadas / S Magnard / E Bettler / J Martin / R Terreux / G Schoehn / C Orelle / J M Jault / P Falson / V Chaptal /
Abstract: Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to translocate substrates out of the cell and detoxify them. While this involves a well-accepted alternating access ...Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to translocate substrates out of the cell and detoxify them. While this involves a well-accepted alternating access mechanism, molecular details of this interplay are still elusive. Rhodamine6G binding on a catalytic inactive mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise michaelian. Here, we investigate this asymmetric behavior via a structural-enzymology approach, solving cryoEM structures of BmrA at defined ATP ratios, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, reveals that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding domains. We observe the same behavior for the other drug Hœchst33342. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the cooperative transition occurs. Altogether, these findings provide a description of the influence of drug binding on the ATP-binding sites through a change in conformational dynamics.
History
DepositionSep 16, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51550.png

Downloads & links

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Map

FileDownload / File: emd_51550.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map by cryosparc v4.5
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 256 pix.
= 214.784 Å		0.84 Å/pix.
x 256 pix.
= 214.784 Å		0.84 Å/pix.
x 256 pix.
= 214.784 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.839 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0923
Minimum - Maximum-0.63821703 - 0.9771102
Average (Standard dev.)0.0020976122 (±0.024687052)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 214.784 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: un-sharpened map

Fileemd_51550_additional_1.map
Annotationun-sharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
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Half map: Half-map A

Fileemd_51550_half_map_1.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_51550_half_map_2.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : BmrA E504A in complex with Hoechst33342

EntireName: BmrA E504A in complex with Hoechst33342
Components
  • Complex: BmrA E504A in complex with Hoechst33342
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
  • Ligand: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE

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Supramolecule #1: BmrA E504A in complex with Hoechst33342

SupramoleculeName: BmrA E504A in complex with Hoechst33342 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 130 KDa

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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
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 65.747141 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSSHHHHHH MPTKKQKSKS KLKPFFALVR RTNPSYGKLA FALALSVVTT LVSLLIPLLT KQLVDGFSMS NLSGTQIGLI ALVFFVQAG LSAYATYALN YNGQKIISGL RELLWKKLIK LPVSYFDTNA SGETVSRVTN DTMVVKELIT THISGFITGI I SVIGSLTI ...String:
MSSSHHHHHH MPTKKQKSKS KLKPFFALVR RTNPSYGKLA FALALSVVTT LVSLLIPLLT KQLVDGFSMS NLSGTQIGLI ALVFFVQAG LSAYATYALN YNGQKIISGL RELLWKKLIK LPVSYFDTNA SGETVSRVTN DTMVVKELIT THISGFITGI I SVIGSLTI LFIMNWKLTL LVLVVVPLAA LILVPIGRKM FSISRETQDE TARFTGLLNQ ILPEIRLVKA SNAEDVEYGR GK MGISSLF KLGVREAKVQ SLVGPLISLV LMAALVAVIG YGGMQVSSGE LTAGALVAFI LYLFQIIMPM GQITTFFTQL QKS IGATER MIEILAEEEE DTVTGKQIEN AHLPIQLDRV SFGYKPDQLI LKEVSAVIEA GKVTAIVGPS GGGKTTLFKL LERF YSPTA GTIRLGDEPV DTYSLESWRE HIGYVSQESP LMSGTIRENI CYGLERDVTD AEIEKAAEMA YALNFIKELP NQFDT EVGE RGIMLSGGQR QRIAIARALL RNPSILMLDA ATSSLDSQSE KSVQQALEVL MEGRTTIVIA HRLSTVVDAD QLLFVE KGE ITGRGTHHEL MASHGLYRDF AEQQLKMNAD LENKAG

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

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Macromolecule #2: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE

MacromoleculeName: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE
type: ligand / ID: 2 / Number of copies: 2 / Formula: HT1
Molecular weightTheoretical: 452.551 Da
Chemical component information

ChemComp-HT1:
2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE

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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

Concentration4 mg/mL
BufferpH: 7.5
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsMembrane protein in detergent

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.5) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: ab-initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 890304
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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