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- EMDB-8469: 3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-8469
Title3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc
Map data3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc
Sample
  • Complex: MsbA reconstituted in lipid nanodisc
    • Protein or peptide: Lipid A export ATP-binding/permease protein MsbA
  • Ligand: PHOSPHATE ION
  • Ligand: 3-HYDROXY-TETRADECANOIC ACID
  • Ligand: MYRISTIC ACID
  • Ligand: LAURIC ACID
KeywordsABC transporter / LPS / flippase / nanodisc / HYDROLASE
Function / homology
Function and homology information


ABC-type lipid A-core oligosaccharide transporter / ABC-type oligopeptide transporter activity / ATPase-coupled lipid transmembrane transporter activity / ATP hydrolysis activity / ATP binding / identical protein binding / 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 (E. coli) / Escherichia coli O157:H7 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsMi W / Walz T
CitationJournal: Nature / Year: 2017
Title: Structural basis of MsbA-mediated lipopolysaccharide transport.
Authors: Wei Mi / Yanyan Li / Sung Hwan Yoon / Robert K Ernst / Thomas Walz / Maofu Liao /
Abstract: Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is ...Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.
History
DepositionNov 8, 2016-
Header (metadata) releaseJan 25, 2017-
Map releaseSep 20, 2017-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tv4
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8469.png

Downloads & links

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Map

FileDownload / File: emd_8469.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 192 pix.
= 236.16 Å		1.23 Å/pix.
x 192 pix.
= 236.16 Å		1.23 Å/pix.
x 192 pix.
= 236.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.10215455 - 0.17202976
Average (Standard dev.)0.000009394499 (±0.011065408)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 236.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z236.160236.160236.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.1020.1720.000

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Supplemental data

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Additional map: 3D cryo-EM reconstruction of nucleotide-free MsbA in lipid...

Fileemd_8469_additional.map
Annotation3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc, without low-pass filter or modification of amplitude
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MsbA reconstituted in lipid nanodisc

EntireName: MsbA reconstituted in lipid nanodisc
Components
  • Complex: MsbA reconstituted in lipid nanodisc
    • Protein or peptide: Lipid A export ATP-binding/permease protein MsbA
  • Ligand: PHOSPHATE ION
  • Ligand: 3-HYDROXY-TETRADECANOIC ACID
  • Ligand: MYRISTIC ACID
  • Ligand: LAURIC ACID

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Supramolecule #1: MsbA reconstituted in lipid nanodisc

SupramoleculeName: MsbA reconstituted in lipid nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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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: Escherichia coli O157:H7 (bacteria)
Molecular weightTheoretical: 67.310445 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGHHHHHHHH HHSSGHIDDD DKHMHNDKDL STWQTFRRLW PTIAPFKAGL IVAGVALILN AASDTFMLSL LKPLLDDGFG KTDRSVLVW MPLVVIGLMI LRGITSYVSS YCISWVSGKV VMTMRRRLFG HMMGMPVSFF DKQSTGTLLS RITYDSEQVA S SSSGALIT ...String:
MGHHHHHHHH HHSSGHIDDD DKHMHNDKDL STWQTFRRLW PTIAPFKAGL IVAGVALILN AASDTFMLSL LKPLLDDGFG KTDRSVLVW MPLVVIGLMI LRGITSYVSS YCISWVSGKV VMTMRRRLFG HMMGMPVSFF DKQSTGTLLS RITYDSEQVA S SSSGALIT VVREGASIIG LFIMMFYYSW QLSIILIVLA PIVSIAIRVV SKRFRNISKN MQNTMGQVTT SAEQMLKGHK EV LIFGGQE VETKRFDKVS NRMRLQGMKM VSASSISDPI IQLIASLALA FVLYAASFPS VMDSLTAGTI TVVFSSMIAL MRP LKSLTN VNAQFQRGMA ACQTLFTILD SEQEKDEGKR VIERATGDVE FRNVTFTYPG RDVPALRNIN LKIPAGKTVA LVGR SGSGK STIASLITRF YDIDEGEILM DGHDLREYTL ASLRNQVALV SQNVHLFNDT VANNIAYART EQYSREQIEE AARMA YAMD FINKMDNGLD TVIGENGVLL SGGQRQRIAI ARALLRDSPI LILDEATSAL DTESERAIQA ALDELQKNRT SLVIAH RLS TIEKADEIVV VEDGVIVERG THNDLLEHRG VYAQLHKMQF GQ

UniProtKB: ATP-dependent lipid A-core flippase

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Macromolecule #3: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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Macromolecule #4: 3-HYDROXY-TETRADECANOIC ACID

MacromoleculeName: 3-HYDROXY-TETRADECANOIC ACID / type: ligand / ID: 4 / Number of copies: 4 / Formula: FTT
Molecular weightTheoretical: 244.37 Da
Chemical component information

ChemComp-FTT:
3-HYDROXY-TETRADECANOIC ACID

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Macromolecule #5: MYRISTIC ACID

MacromoleculeName: MYRISTIC ACID / type: ligand / ID: 5 / Number of copies: 1 / Formula: MYR
Molecular weightTheoretical: 228.371 Da
Chemical component information

ChemComp-MYR:
MYRISTIC ACID

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Macromolecule #6: LAURIC ACID

MacromoleculeName: LAURIC ACID / type: ligand / ID: 6 / Number of copies: 1 / Formula: DAO
Molecular weightTheoretical: 200.318 Da
Chemical component information

ChemComp-DAO:
LAURIC ACID

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Cylinder density
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 67220
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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