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- EMDB-13095: CryoEM structure of the ABC transporter BmrA E504A mutant in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-13095
TitleCryoEM structure of the ABC transporter BmrA E504A mutant in complex with ATP-Mg
Map dataSharpened map
Sample
  • Complex: Complex of BmrA with ATP-Mg
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
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) / Bacillus subtilis (strain 168) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGobet A / Schoehn G / Falson P / Chaptal V
Funding support France, 6 items
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, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 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: 0.391
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.391
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7ow8
  • Surface level: 0.391
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13095.png

Downloads & links

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Map

FileDownload / File: emd_13095.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.052 Å
Density
Contour LevelBy AUTHOR: 0.391 / Movie #1: 0.391
Minimum - Maximum-2.9704797 - 4.346474
Average (Standard dev.)0.0009915512 (±0.07646729)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 269.312 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0521.0521.052
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z269.312269.312269.312
α/β/γ90.00090.00090.000
start NX/NY/NZ258279248
NX/NY/NZ8855101
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.9704.3460.001

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Supplemental data

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Additional map: Unsharpened map

Fileemd_13095_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: even half map, unsharpened

Fileemd_13095_half_map_1.map
Annotationeven half map, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: odd half map, unsharpened

Fileemd_13095_half_map_2.map
Annotationodd half map, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of BmrA with ATP-Mg

EntireName: Complex of BmrA with ATP-Mg
Components
  • Complex: Complex of BmrA with ATP-Mg
    • Protein or peptide: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of BmrA with ATP-Mg

SupramoleculeName: Complex of BmrA with ATP-Mg / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168
Recombinant expressionOrganism: Escherichia coli (E. coli)
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
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Bacillus subtilis (strain 168) (bacteria) / Strain: 168
Molecular weightTheoretical: 65.489824 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 LEN

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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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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
Component:
ConcentrationFormulaName
20.0 mMHepeshepes
100.0 mMNaClSodium chlorideNaClSodium chloride
0.035 % (W/V)DDMDDM
0.03 % (W/V)CholateCholic acidCholateCholic acid
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 4400 / Average exposure time: 5.0 sec. / Average electron dose: 37.95 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 3.2)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6r81

Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Software - details: Non Uniform refinement / Number images used: 327764

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Atomic model buiding 1

Initial modelPDB ID:

6r81


Chain - Chain ID: A / Chain - Residue range: 1-600
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7ow8:
CryoEM structure of the ABC transporter BmrA E504A mutant in complex with ATP-Mg

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