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- EMDB-12170: Multidrug resistance transporter BmrA mutant E504A bound with ATP... -

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Basic information

Entry
Database: EMDB / ID: EMD-12170
TitleMultidrug resistance transporter BmrA mutant E504A bound with ATP, Mg, and Rhodamine 6G solved by Cryo-EM
Map dataMain map used for model building.
Sample
  • Complex: Multidrug resistance transporter from Bacillus subtilis bound with ATP, Mg, and Rhodamine 6G.
    • Protein or peptide: Lipid A export ATP-binding/permease protein MsbA
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: RHODAMINE 6G
Function / homology
Function and homology information


ABC-type transporter activity / ATP hydrolysis activity / ATP binding / 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
Lipid A export ATP-binding/permease protein MsbA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWiseman B / Chaptal V / Zampieri V / Magnard S / Hogbom M / Falson P
Funding support France, Sweden, 3 items
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, 2021-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bg4
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bg4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12170.png

Downloads & links

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Map

FileDownload / File: emd_12170.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map used for model building.
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.24 / Movie #1: 0.27
Minimum - Maximum-0.6137139 - 1.2895635
Average (Standard dev.)0.010118798 (±0.057938572)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.6141.2900.010

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Supplemental data

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Additional map: Sharpened map used to aid and improve model building in many areas

Fileemd_12170_additional_1.map
AnnotationSharpened map used to aid and improve model building in many areas
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Multidrug resistance transporter from Bacillus subtilis bound wit...

EntireName: Multidrug resistance transporter from Bacillus subtilis bound with ATP, Mg, and Rhodamine 6G.
Components
  • Complex: Multidrug resistance transporter from Bacillus subtilis bound with ATP, Mg, and Rhodamine 6G.
    • Protein or peptide: Lipid A export ATP-binding/permease protein MsbA
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: RHODAMINE 6G

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Supramolecule #1: Multidrug resistance transporter from Bacillus subtilis bound wit...

SupramoleculeName: Multidrug resistance transporter from Bacillus subtilis bound with ATP, Mg, and Rhodamine 6G.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: mutant E504A
Source (natural)Organism: Bacillus subtilis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C43, delta-AcrB / Recombinant plasmid: pET15b

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Macromolecule #1: Lipid A export ATP-binding/permease protein MsbA

MacromoleculeName: Lipid A export ATP-binding/permease protein MsbA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
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

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: RHODAMINE 6G

MacromoleculeName: RHODAMINE 6G / type: ligand / ID: 4 / Number of copies: 2 / Formula: RHQ
Molecular weightTheoretical: 443.557 Da
Chemical component information

ChemComp-R6G:
RHODAMINE 6G / Rhodamine 6G

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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

Concentration3.5 mg/mL
BufferpH: 8
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3477 / Average exposure time: 5.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 486404
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v2) / Number images used: 128372
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 187
Output model

PDB-7bg4:
Multidrug resistance transporter BmrA mutant E504A bound with ATP, Mg, and Rhodamine 6G solved by Cryo-EM

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