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- PDB-8ri1: BmrA E504-100uMATPMg -

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Basic information

Entry
Database: PDB / ID: 8ri1
TitleBmrA E504-100uMATPMg
ComponentsMultidrug resistance ABC transporter ATP-binding/permease protein BmrA
KeywordsTRANSPORT PROTEIN / BmrA / multidrug transporter / drug resistance / ABC transporter
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
ADENOSINE-5'-TRIPHOSPHATE / Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsGobet, A. / Zarkadas, E. / Schoehn, G. / Falson, P. / Chaptal, V.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0023-01 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
DepositionDec 18, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
B: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,5576
Polymers131,4942
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 65747.141 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: bmrA, yvcC, BSU34820 / Production host: Escherichia coli (E. coli)
References: UniProt: O06967, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Type: COMPLEX / Details: Apo form, mutant E504A / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Cell: C43 (Delta AcrB) / Plasmid: pET15b
Buffer solutionpH: 7.5
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3218

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7Cootmodel fitting
8ISOLDEmodel fitting
10PHENIXmodel refinement
14cryoSPARC3D reconstructionNU refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3787631
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 378688 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6r81

6r81


Pdb chain-ID: AB / Accession code: 6r81 / Chain residue range: 1-580 / Pdb chain residue range: 1-580 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0119034
ELECTRON MICROSCOPYf_angle_d1.25712258
ELECTRON MICROSCOPYf_dihedral_angle_d4.9631234
ELECTRON MICROSCOPYf_chiral_restr0.1171490
ELECTRON MICROSCOPYf_plane_restr0.0051516

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