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- PDB-7ow8: CryoEM structure of the ABC transporter BmrA E504A mutant in comp... -

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Basic information

Entry
Database: PDB / ID: 7ow8
TitleCryoEM structure of the ABC transporter BmrA E504A mutant in complex with ATP-Mg
ComponentsMultidrug resistance ABC transporter ATP-binding/permease protein BmrA
KeywordsTRANSPORT PROTEIN / BmrA ABC transporter complex with ATP-Mg 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 / Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGobet, A. / Schoehn, G. / Falson, P. / Chaptal, V.
Funding support France, 6items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-CLAMP- 13-BSV5-0001-01 France
Agence Nationale de la Recherche (ANR)ANR-NMX-14-CE09-0024-03 France
Agence Nationale de la Recherche (ANR)ANR-CAVEOTANK-17-CE11-0015-03 France
Agence Nationale de la Recherche (ANR)ANR-CLAMP2- 18-CE11-0002-01 France
Agence Nationale de la Recherche (ANR)ANR-19-CE11- 0023-01 France
French League Against Cancercomite Ardeche 212471 France
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
DepositionJun 17, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release
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Revision 1.1Feb 2, 2022Group: Database references / Category: citation / citation_author
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Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
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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)132,0436
Polymers130,9802
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, All structures of ABC proteins Activity assays
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16320 Å2
ΔGint-112 kcal/mol
Surface area48610 Å2

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Components

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


Mass: 65489.824 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Strain: 168 / 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 / Feature type: SUBJECT OF INVESTIGATION / 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 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: Complex of BmrA with ATP-Mg / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMhepesHepes1
2100 mMNaClNaCl1
30.035 % (W/V)DDMDDM1
40.03 % (W/V)CholateCholate1
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: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Cs: 2.7 mm
Image recordingAverage exposure time: 5 sec. / Electron dose: 37.95 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4400

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4cryoSPARC3.2CTF correction
7Coot0.9.4model fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstructionNon Uniform refinement
13ISOLDE1.1model refinement
14PHENIXmodel refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 327764 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6R81

6r81


Pdb chain-ID: A / Accession code: 6R81 / Pdb chain residue range: 1-600 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0099114
ELECTRON MICROSCOPYf_angle_d1.14512348
ELECTRON MICROSCOPYf_dihedral_angle_d9.7285504
ELECTRON MICROSCOPYf_chiral_restr0.0641488
ELECTRON MICROSCOPYf_plane_restr0.0091542

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