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- PDB-7bg4: Multidrug resistance transporter BmrA mutant E504A bound with ATP... -

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Basic information

Entry
Database: PDB / ID: 7bg4
TitleMultidrug resistance transporter BmrA mutant E504A bound with ATP, Mg, and Rhodamine 6G solved by Cryo-EM
ComponentsMultidrug resistance ABC transporter ATP-binding/permease protein BmrA
KeywordsTRANSPORT PROTEIN / membrane protein / ABC transporter / multidrug 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
ADENOSINE-5'-TRIPHOSPHATE / RHODAMINE 6G / Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWiseman, B. / Chaptal, V. / Zampieri, V. / Magnard, S. / Hogbom, M. / Falson, P.
Funding support France, Sweden, 3items
OrganizationGrant numberCountry
French National Research AgencyANR-17-CE11-0015-03 France
Knut and Alice Wallenberg Foundation Sweden
Swedish Research Council Sweden
CitationJournal: Sci Adv / Year: 2022
Title: Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter.
Authors: Vincent Chaptal / Veronica Zampieri / Benjamin Wiseman / Cédric Orelle / Juliette Martin / Kim-Anh Nguyen / Alexia Gobet / Margot Di Cesare / Sandrine Magnard / Waqas Javed / Jad Eid / ...Authors: Vincent Chaptal / Veronica Zampieri / Benjamin Wiseman / Cédric Orelle / Juliette Martin / Kim-Anh Nguyen / Alexia Gobet / Margot Di Cesare / Sandrine Magnard / Waqas Javed / Jad Eid / Arnaud Kilburg / Marine Peuchmaur / Julien Marcoux / Luca Monticelli / Martin Hogbom / Guy Schoehn / Jean-Michel Jault / Ahcène Boumendjel / Pierre Falson /
Abstract: Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg-bound outward-facing ...Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg-bound outward-facing conformations of the (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in . Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.
History
DepositionJan 5, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Advisory / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / pdbx_validate_close_contact / struct_conn / struct_conn_type
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
Revision 1.2Jul 10, 2024Group: Data collection / Database references ...Data collection / Database references / Source and taxonomy / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / entity / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _em_admin.last_update / _entity.pdbx_description ..._em_admin.last_update / _entity.pdbx_description / _entity.pdbx_ec / _entity_src_gen.pdbx_gene_src_gene / _struct_ref.db_code / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq_dif.pdbx_seq_db_accession_code

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Structure visualization

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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)133,4448
Polymers131,4942
Non-polymers1,9506
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17950 Å2
ΔGint-114 kcal/mol
Surface area51510 Å2
MethodPISA

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Components

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


Mass: 65747.141 Da / Num. of mol.: 2 / Mutation: E504A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: bmrA, yvcC, BSU34820 / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Variant (production host): C43
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
#4: Chemical ChemComp-RHQ / RHODAMINE 6G


Mass: 443.557 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C28H31N2O3
Has ligand of interestN

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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 transporter from Bacillus subtilis bound with ATP, Mg, and Rhodamine 6G.
Type: COMPLEX / Details: mutant E504A / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43, delta-AcrB / Plasmid: pET15b
Buffer solutionpH: 8
SpecimenConc.: 3.5 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: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3477
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv2particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11cryoSPARCv2initial Euler assignment
12cryoSPARCv2final Euler assignment
13cryoSPARCv2classification
14cryoSPARCv23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 486404
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128372 / Symmetry type: POINT
Atomic model buildingB value: 187 / Protocol: FLEXIBLE FIT / Space: REAL

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