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- EMDB-8669: CryoEM map of MsbA in POPG nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-8669
TitleCryoEM map of MsbA in POPG nanodisc
Map datamap of MsbA with LPS in POPG nanodisc, filtered to 5A and sharpened with -280 B-factor
Sample
  • Complex: MsbA with LPS in POPG nanodisc
    • Protein or peptide: MsbA
Function / homology
Function and homology information


ABC-type lipid A-core oligosaccharide transporter / ATPase-coupled lipid transmembrane transporter activity / ABC-type 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsMi W / Walz T / Liao M
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
DepositionApr 11, 2017-
Header (metadata) releaseJul 19, 2017-
Map releaseSep 20, 2017-
UpdateSep 27, 2017-
Current statusSep 27, 2017Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8669.png

Downloads & links

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Map

FileDownload / File: emd_8669.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of MsbA with LPS in POPG nanodisc, filtered to 5A and sharpened with -280 B-factor
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.28 Å/pix.
x 192 pix.
= 245.76 Å		1.28 Å/pix.
x 192 pix.
= 245.76 Å		1.28 Å/pix.
x 192 pix.
= 245.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.034 / Movie #1: 0.04
Minimum - Maximum-0.06834029 - 0.14602281
Average (Standard dev.)0.000037362337 (±0.00919677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 245.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z245.760245.760245.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.0680.1460.000

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Supplemental data

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Additional map: map without any filter or bfactor sharpen

Fileemd_8669_additional.map
Annotationmap without any filter or bfactor sharpen
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 with LPS in POPG nanodisc

EntireName: MsbA with LPS in POPG nanodisc
Components
  • Complex: MsbA with LPS in POPG nanodisc
    • Protein or peptide: MsbA

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Supramolecule #1: MsbA with LPS in POPG nanodisc

SupramoleculeName: MsbA with LPS in POPG nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 67.2 KDa

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Macromolecule #1: MsbA

MacromoleculeName: MsbA / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGHHHHHHHH HHSSGHIDDD DKHMHNDKDL STWQTFRRLW PTIAPFKAGL IVAGVALILN AASDTFMLSL LKPLLDDGFG KTDRSVLVWM PLVVIGLMIL RGITSYVSSY CISWVSGKVV MTMRRRLFGH MMGMPVSFFD KQSTGTLLSR ITYDSEQVAS SSSGALITVV ...String:
MGHHHHHHHH HHSSGHIDDD DKHMHNDKDL STWQTFRRLW PTIAPFKAGL IVAGVALILN AASDTFMLSL LKPLLDDGFG KTDRSVLVWM PLVVIGLMIL RGITSYVSSY CISWVSGKVV MTMRRRLFGH MMGMPVSFFD KQSTGTLLSR ITYDSEQVAS SSSGALITVV REGASIIGLF IMMFYYSWQL SIILIVLAPI VSIAIRVVSK RFRNISKNMQ NTMGQVTTSA EQMLKGHKEV LIFGGQEVET KRFDKVSNRM RLQGMKMVSA SSISDPIIQL IASLALAFVL YAASFPSVMD SLTAGTITVV FSSMIALMRP LKSLTNVNAQ FQRGMAACQT LFTILDSEQE KDEGKRVIER ATGDVEFRNV TFTYPGRDVP ALRNINLKIP AGKTVALVGR SGSGKSTIAS LITRFYDIDE GEILMDGHDL REYTLASLRN QVALVSQNVH LFNDTVANNI AYARTEQYSR EQIEEAARMA YAMDFINKMD NGLDTVIGEN GVLLSGGQRQ RIAIARALLR DSPILILDEA TSALDTESER AIQAALDELQ KNRTSLVIAH RLSTIEKADE IVVVEDGVIV ERGTHNDLLE HRGVYAQLHK MQFGQ

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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

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 7.2 sec. / 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

Startup modelType of model: EMDB MAP
EMDB ID:

8469

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 55199
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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