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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: 8469
Title3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc
Map data3D cryo-EM reconstruction of nucleotide-free MsbA in lipid nanodisc
SampleMsbA reconstituted in lipid nanodisc
  • Lipid A export ATP-binding/permease protein MsbA
  • (ligand) x 7
Function / homologyABC transporter, conserved site / ABC transporters family signature. / Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter-like / AAA+ ATPase domain / ABC transporter type 1, transmembrane domain / ABC transporter, lipid A export, MsbA / ATP-binding cassette, ABC transporter-type domain profile. / P-loop containing nucleoside triphosphate hydrolase ...ABC transporter, conserved site / ABC transporters family signature. / Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter-like / AAA+ ATPase domain / ABC transporter type 1, transmembrane domain / ABC transporter, lipid A export, MsbA / ATP-binding cassette, ABC transporter-type domain profile. / P-loop containing nucleoside triphosphate hydrolase / ABC transporter type 1, transmembrane domain superfamily / ABC transporter / ABC transporter transmembrane region / lipid-transporting ATPase activity / ec:3.6.3.-: / integral component of membrane / ATP binding / plasma membrane / Lipid A export ATP-binding/permease protein MsbA
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 4.2 Å resolution
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.
Validation ReportPDB-ID: 5tv4

SummaryFull reportAbout validation report
DateDeposition: Nov 8, 2016 / Header (metadata) release: Jan 25, 2017 / Map release: Sep 20, 2017 / Last update: Nov 8, 2017

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Structure visualization

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

Downloads & links

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Map

Fileemd_8469.map.gz (map file in CCP4 format, 28312 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
192 pix
1.23 Å/pix.
= 236.16 Å
192 pix
1.23 Å/pix.
= 236.16 Å
192 pix
1.23 Å/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
Contour Level:0.045 (by author), 0.045 (movie #1):
Minimum - Maximum-0.10215455 - 0.17202976
Average (Standard dev.)9.394499E-6 (0.011065408)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions192192192
Origin-96-96-96
Limit959595
Spacing192192192
CellA=B=C: 236.16 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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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Sample components

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Entire MsbA reconstituted in lipid nanodisc

EntireName: MsbA reconstituted in lipid nanodisc / Number of components: 9

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Component #1: protein, MsbA reconstituted in lipid nanodisc

ProteinName: MsbA reconstituted in lipid nanodisc / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Vector: pET-19a

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Component #2: protein, Lipid A export ATP-binding/permease protein MsbA

ProteinName: Lipid A export ATP-binding/permease protein MsbA / Recombinant expression: No
MassTheoretical: 67.310445 kDa
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #3: ligand, 2-amino-2-deoxy-alpha-D-glucopyranose

LigandName: 2-amino-2-deoxy-alpha-D-glucopyranose / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.179171 kDa

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Component #4: ligand, 3-DEOXY-D-MANNO-OCT-2-ULOSONIC ACID

LigandName: 3-DEOXY-D-MANNO-OCT-2-ULOSONIC ACID / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.238192 kDa

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Component #5: ligand, L-GLYCERO-D-MANNO-HEPTOPYRANOSE

LigandName: L-GLYCERO-D-MANNO-HEPTOPYRANOSE / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.210182 kDa

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Component #6: ligand, PHOSPHATE ION

LigandName: PHOSPHATE IONPhosphate / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 9.497105 MDa

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Component #7: ligand, 3-HYDROXY-TETRADECANOIC ACID

LigandName: 3-HYDROXY-TETRADECANOIC ACID / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.24437 kDa

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Component #8: ligand, MYRISTIC ACID

LigandName: MYRISTIC ACID / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.228371 kDa

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Component #9: ligand, LAURIC ACID

LigandName: LAURIC ACID / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.200318 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 47 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 67220
3D reconstructionSoftware: RELION / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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