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- EMDB-12145: Structure of MsbA in Salipro with ADP vanadate -

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Basic information

Entry
Database: EMDB / ID: EMD-12145
TitleStructure of MsbA in Salipro with ADP vanadate
Map data
Sample
  • Complex: MsbA in Salipro with ADP vanadate
    • Protein or peptide: ATP-dependent lipid A-core flippase
  • Ligand: VANADATE ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
KeywordsLipid export MsbA / MEMBRANE PROTEIN
Function / homology
Function and homology information


MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ATP-binding cassette (ABC) transporter complex / transmembrane transport ...MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ATP-binding cassette (ABC) transporter complex / transmembrane transport / lipid binding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter, lipid A-core flippase, MsbA / 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 ...Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter, lipid A-core flippase, MsbA / 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
ATP-dependent lipid A-core flippase
Similarity search - Component
Biological speciesEscherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsTraore DAK / Tidow H
CitationJournal: FEBS J / Year: 2022
Title: Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination.
Authors: Dominique-Maurice Kehlenbeck / Daouda A K Traore / Inokentijs Josts / Simon Sander / Martine Moulin / Michael Haertlein / Sylvain Prevost / V Trevor Forsyth / Henning Tidow /
Abstract: The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative ...The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins.
History
DepositionDec 21, 2020-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bcw
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12145.png

Downloads & links

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Map

FileDownload / File: emd_12145.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.827 Å
Density
Contour LevelBy AUTHOR: 0.325 / Movie #1: 0.325
Minimum - Maximum-0.7483251 - 1.2516495
Average (Standard dev.)0.0026060438 (±0.04078053)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8270.8270.827
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z264.640264.640264.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.7481.2520.003

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Supplemental data

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Sample components

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Entire : MsbA in Salipro with ADP vanadate

EntireName: MsbA in Salipro with ADP vanadate
Components
  • Complex: MsbA in Salipro with ADP vanadate
    • Protein or peptide: ATP-dependent lipid A-core flippase
  • Ligand: VANADATE ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate

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Supramolecule #1: MsbA in Salipro with ADP vanadate

SupramoleculeName: MsbA in Salipro with ADP vanadate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)

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Macromolecule #1: ATP-dependent lipid A-core flippase

MacromoleculeName: ATP-dependent lipid A-core flippase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: ABC-type lipid A-core oligosaccharide transporter
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 66.486664 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHEN LYFQGSMHND KDLSTWQTFR RLWPTIAPFK AGLIVAGVAL ILNAASDTFM LSLLKPLLDD GFGKTDRSVL VWMPLVVIG LMILRGITSY VSSYCISWVS GKVVMTMRRR LFGHMMGMPV SFFDKQSTGT LLSRITYDSE QVASSSSGAL I TVVREGAS ...String:
MGHHHHHHEN LYFQGSMHND KDLSTWQTFR RLWPTIAPFK AGLIVAGVAL ILNAASDTFM LSLLKPLLDD GFGKTDRSVL VWMPLVVIG LMILRGITSY VSSYCISWVS GKVVMTMRRR LFGHMMGMPV SFFDKQSTGT LLSRITYDSE QVASSSSGAL I TVVREGAS IIGLFIMMFY YSWQLSIILI VLAPIVSIAI RVVSKRFRNI SKNMQNTMGQ VTTSAEQMLK GHKEVLIFGG QE VETKRFD KVSNRMRLQG MKMVSASSIS DPIIQLIASL ALAFVLYAAS FPSVMDSLTA GTITVVFSSM IALMRPLKSL TNV NAQFQR GMAACQTLFT ILDSEQEKDE GKRVIERATG DVEFRNVTFT YPGRDVPALR NINLKIPAGK TVALVGRSGS GKST IASLI TRFYDIDEGE ILMDGHDLRE YTLASLRNQV ALVSQNVHLF NDTVANNIAY ARTEQYSREQ IEEAARMAYA MDFIN KMDN GLDTVIGENG VLLSGGQRQR IAIARALLRD SPILILDEAT SALDTESERA IQAALDELQK NRTSLVIAHR LSTIEK ADE IVVVEDGVIV ERGTHNDLLE HRGVYAQLHK MQFGQ

UniProtKB: ATP-dependent lipid A-core flippase

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Macromolecule #2: VANADATE ION

MacromoleculeName: VANADATE ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: VO4
Molecular weightTheoretical: 114.939 Da
Chemical component information

ChemComp-VN3:
VANADATE ION

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 5 / Number of copies: 2 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM

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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

Concentration0.6 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
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. 2.15) / Number images used: 83278
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5ttp


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7bcw:
Structure of MsbA in Salipro with ADP vanadate

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