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- EMDB-8670: CryoEM map of MsbA with lipid A in POPG nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-8670
TitleCryoEM map of MsbA with lipid A in POPG nanodisc
Map dataMsbA with lipid A in POPG nanodisc filtered to 4.5 A and with -220 B-factor sharpening
Sample
  • Complex: MsbA with lipid A in POPG nanodisc
    • Protein or peptide: MsbA with lipid A in POPG nanodisc
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: 4.5 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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8670.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMsbA with lipid A in POPG nanodisc filtered to 4.5 A and with -220 B-factor sharpening
Voxel sizeX=Y=Z: 1.28 Å
Density
Contour LevelBy AUTHOR: 0.0235 / Movie #1: 0.04
Minimum - Maximum-0.061054986 - 0.14212495
Average (Standard dev.)0.000107734224 (±0.008882276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 204.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z204.800204.800204.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-0.0610.1420.000

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Supplemental data

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Additional map: Map with no filtering or sharpening

Fileemd_8670_additional.map
AnnotationMap with no filtering or sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : MsbA with lipid A in POPG nanodisc

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

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Supramolecule #1: MsbA with lipid A in POPG nanodisc

SupramoleculeName: MsbA with lipid A in POPG nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: MsbA expressed from special E.coli strain ClearColi (which has no LPS), mixed with lipid A and reconstituted into POPG nanodisc
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Recombinant strain: ClearColi

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Macromolecule #1: MsbA with lipid A in POPG nanodisc

MacromoleculeName: MsbA with lipid A in POPG nanodisc / 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)
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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average exposure time: 7.2 sec. / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 554677
Startup modelType of model: EMDB MAP
EMDB ID:
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 85496
FSC plot (resolution estimation)

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