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- EMDB-13404: Nanodisc reconstituted MsbA in complex with nanobodies, spin-labe... -

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Entry
Database: EMDB / ID: EMD-13404
TitleNanodisc reconstituted MsbA in complex with nanobodies, spin-labeled at position A60C
Map data
Sample
  • Complex: E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at Cys60
    • Protein or peptide: ATP-dependent lipid A-core flippase
    • Protein or peptide: Nb_MsbA#1
  • Ligand: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{R},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{S},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-3,4,5-tris(oxidanyl)oxan-2-yl]oxy-3,5-bis(oxidanyl)oxan-2-yl]oxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-4-[(3~{R})-3-nonanoyloxytetradecanoyl]oxy-5-[[(3~{R})-3-octanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{S},5~{S},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanylnonanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-oxan-2-yl]methoxy]-5-oxidanyl-oxane-2-carboxylic acid
  • Ligand: (1~{R},4~{R},11~{S},14~{S},19~{Z})-19-[2-[2,5-bis(oxidanylidene)pyrrolidin-1-yl]ethylimino]-7,8,17,18-tetraoxa-1,4,11,14-tetrazatricyclo[12.6.2.2^{4,11}]tetracosane-6,9,16-trione
  • Ligand: GADOLINIUM ATOM
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 / membrane / 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 K-12 (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsJanuliene D / Parey K / Galazzo L / Meier G / Vecchis D / Striednig B / Hilbi H / Schaefer LV / Kuprov I / Bordignon E ...Januliene D / Parey K / Galazzo L / Meier G / Vecchis D / Striednig B / Hilbi H / Schaefer LV / Kuprov I / Bordignon E / Seeger MA / Moeller A
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)CRC944 Germany
German Research Foundation (DFG)SCHA 1574/6-1 Germany
German Research Foundation (DFG)EXC 2033 - 390677874 - RESOLV Germany
CitationJournal: Sci Adv / Year: 2022
Title: The ABC transporter MsbA adopts the wide inward-open conformation in cells.
Authors: Laura Galazzo / Gianmarco Meier / Dovile Januliene / Kristian Parey / Dario De Vecchis / Bianca Striednig / Hubert Hilbi / Lars V Schäfer / Ilya Kuprov / Arne Moeller / Enrica Bordignon / Markus A Seeger /
Abstract: Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their ...Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.
History
DepositionAug 16, 2021-
Header (metadata) releaseAug 24, 2022-
Map releaseAug 24, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13404.png

Downloads & links

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Map

FileDownload / File: emd_13404.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.837 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.4329513 - 1.8225129
Average (Standard dev.)0.0019300686 (±0.045619432)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 267.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13404_msk_1.map
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Additional map: Locally filtered map, used for figures and assisted model building

Fileemd_13404_additional_1.map
AnnotationLocally filtered map, used for figures and assisted model building
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Additional map: #1

Fileemd_13404_additional_2.map
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Half map: #2

Fileemd_13404_half_map_1.map
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Half map: #1

Fileemd_13404_half_map_2.map
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Sample components

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Entire : E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-...

EntireName: E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at Cys60
Components
  • Complex: E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at Cys60
    • Protein or peptide: ATP-dependent lipid A-core flippase
    • Protein or peptide: Nb_MsbA#1
  • Ligand: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{R},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{S},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-3,4,5-tris(oxidanyl)oxan-2-yl]oxy-3,5-bis(oxidanyl)oxan-2-yl]oxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-4-[(3~{R})-3-nonanoyloxytetradecanoyl]oxy-5-[[(3~{R})-3-octanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{S},5~{S},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanylnonanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-oxan-2-yl]methoxy]-5-oxidanyl-oxane-2-carboxylic acid
  • Ligand: (1~{R},4~{R},11~{S},14~{S},19~{Z})-19-[2-[2,5-bis(oxidanylidene)pyrrolidin-1-yl]ethylimino]-7,8,17,18-tetraoxa-1,4,11,14-tetrazatricyclo[12.6.2.2^{4,11}]tetracosane-6,9,16-trione
  • Ligand: GADOLINIUM ATOM

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Supramolecule #1: E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-...

SupramoleculeName: E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at Cys60
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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 K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 65.760852 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPDEAEKLFN QMHNDKDLST WQTFRRLWPT IAPFKAGLIV AGVALILNAA SDTFMLSLLK PLLDDGFGKT DRSVLVWMPL VVIGLMILR GITSYVSSYC ISWVSGKVVM TMRRRLFGHM MGMPVSFFDK QSTGTLLSRI TYDSEQVASS SSGALITVVR E GASIIGLF ...String:
GPDEAEKLFN QMHNDKDLST WQTFRRLWPT IAPFKAGLIV AGVALILNAA SDTFMLSLLK PLLDDGFGKT DRSVLVWMPL VVIGLMILR GITSYVSSYC ISWVSGKVVM TMRRRLFGHM MGMPVSFFDK QSTGTLLSRI TYDSEQVASS SSGALITVVR E GASIIGLF IMMFYYSWQL SIILIVLAPI VSIAIRVVSK RFRNISKNMQ NTMGQVTTSA EQMLKGHKEV LIFGGQEVET KR FDKVSNR MRLQGMKMVS ASSISDPIIQ LIASLALAFV LYAASFPSVM DSLTAGTITV VFSSMIALMR PLKSLTNVNA QFQ RGMAAC QTLFTILDSE QEKDEGKRVI ERATGDVEFR NVTFTYPGRD VPALRNINLK IPAGKTVALV GRSGSGKSTI ASLI TRFYD IDEGEILMDG HDLREYTLAS LRNQVALVSQ NVHLFNDTVA NNIAYARTEQ YSREQIEEAA RMAYAMDFIN KMDNG LDTV IGENGVLLSG GQRQRIAIAR ALLRDSPILI LDEATSALDT ESERAIQAAL DELQKNRTSL VIAHRLSTIE KADEIV VVE DGVIVERGTH NDLLEHRGVY AQLHKMQFGQ

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Macromolecule #2: Nb_MsbA#1

MacromoleculeName: Nb_MsbA#1 / type: protein_or_peptide / ID: 2 / Details: Nanobody is labeled with GMO-GD at Cys60 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 12.369879 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GPSQMQLVES GGGLVQAGGS LRLSCAVSGS IFSIITLAWY RQAPGKPREN VATITRGSRT SYCDSVKGRF TISKDNAKST VYLQMNKLK PEDTADYYCN AEGPAGYWGQ GTPVTVS

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Macromolecule #3: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4-[(...

MacromoleculeName: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{R},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{S},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl) ...Name: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{R},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{S},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-3,4,5-tris(oxidanyl)oxan-2-yl]oxy-3,5-bis(oxidanyl)oxan-2-yl]oxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-4-[(3~{R})-3-nonanoyloxytetradecanoyl]oxy-5-[[(3~{R})-3-octanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{S},5~{S},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanylnonanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-oxan-2-yl]methoxy]-5-oxidanyl-oxane-2-carboxylic acid
type: ligand / ID: 3 / Number of copies: 1 / Formula: EIW
Molecular weightTheoretical: 2.206503 KDa
Chemical component information

ChemComp-EIW:
(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{R},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-4-[(2~{R},3~{S},4~{S},5~{S},6~{R})-6-[(1~{S})-1,2-bis(oxidanyl)ethyl]-3,4,5-tris(oxidanyl)oxan-2-yl]oxy-3,5-bis(oxidanyl)oxan-2-yl]oxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-4-[(3~{R})-3-nonanoyloxytetradecanoyl]oxy-5-[[(3~{R})-3-octanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{S},5~{S},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanylnonanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-oxan-2-yl]methoxy]-5-oxidanyl-oxane-2-carboxylic acid

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Macromolecule #4: (1~{R},4~{R},11~{S},14~{S},19~{Z})-19-[2-[2,5-bis(oxidanylidene)p...

MacromoleculeName: (1~{R},4~{R},11~{S},14~{S},19~{Z})-19-[2-[2,5-bis(oxidanylidene)pyrrolidin-1-yl]ethylimino]-7,8,17,18-tetraoxa-1,4,11,14-tetrazatricyclo[12.6.2.2^{4,11}]tetracosane-6,9,16-trione
type: ligand / ID: 4 / Number of copies: 2 / Formula: 88T
Molecular weightTheoretical: 524.524 Da
Chemical component information

ChemComp-88T:
(1~{R},4~{R},11~{S},14~{S},19~{Z})-19-[2-[2,5-bis(oxidanylidene)pyrrolidin-1-yl]ethylimino]-7,8,17,18-tetraoxa-1,4,11,14-tetrazatricyclo[12.6.2.2^{4,11}]tetracosane-6,9,16-trione

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Macromolecule #5: GADOLINIUM ATOM

MacromoleculeName: GADOLINIUM ATOM / type: ligand / ID: 5 / Number of copies: 2 / Formula: GD
Molecular weightTheoretical: 157.25 Da
Chemical component information

ChemComp-GD:
GADOLINIUM ATOM

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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

Concentration2.6 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.0.1) / Number images used: 142832
FSC plot (resolution estimation)

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