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- EMDB-13406: AMP-PNP bound nanodisc reconstituted MsbA with nanobodies, spin-l... -

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Basic information

Entry
Database: EMDB / ID: EMD-13406
TitleAMP-PNP bound nanodisc reconstituted MsbA with nanobodies, spin-labeled at position T68C
Map data
Sample
  • Complex: AMP-PNP bound E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at T68C
    • Protein or peptide: ATP-dependent lipid A-core flippase
    • Protein or peptide: Nanobody Nb_MsbA#1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • 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
  • Ligand: water
KeywordsABC transporter / nanobody / AMP-PNP / Gd-DOTA / MEMBRANE PROTEIN
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 / identical protein binding / membrane / 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 (strain K12) (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 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-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13406.png

Downloads & links

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Map

FileDownload / File: emd_13406.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 320 pix.
= 267.84 Å		0.84 Å/pix.
x 320 pix.
= 267.84 Å		0.84 Å/pix.
x 320 pix.
= 267.84 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.837 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-3.9757144 - 7.89592
Average (Standard dev.)0.0018979686 (±0.118374996)
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_13406_msk_1.map
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Additional map: Locally filtered map, used for figures and assisted model building

Fileemd_13406_additional_1.map
AnnotationLocally filtered map, used for figures and assisted model building
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Additional map: Density modified map, used for model building

Fileemd_13406_additional_2.map
AnnotationDensity modified map, used for model building
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Half map: #2

Fileemd_13406_half_map_1.map
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Half map: #1

Fileemd_13406_half_map_2.map
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Sample components

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Entire : AMP-PNP bound E. coli MsbA in complex with nanobody Nb_MsbA#1, la...

EntireName: AMP-PNP bound E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at T68C
Components
  • Complex: AMP-PNP bound E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at T68C
    • Protein or peptide: ATP-dependent lipid A-core flippase
    • Protein or peptide: Nanobody Nb_MsbA#1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • 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
  • Ligand: water

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Supramolecule #1: AMP-PNP bound E. coli MsbA in complex with nanobody Nb_MsbA#1, la...

SupramoleculeName: AMP-PNP bound E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at T68C
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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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 (strain K12) (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

UniProtKB: ATP-dependent lipid A-core flippase

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Macromolecule #2: Nanobody Nb_MsbA#1

MacromoleculeName: Nanobody Nb_MsbA#1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 12.41093 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GPSQMQLVES GGGLVQAGGS LRLSCAVSGS IFSIITLAWY RQAPGKPREN VATITRGSRT SYADSVKGRF CISKDNAKST VYLQMNKLK PEDTADYYCN AEGPAGYWGQ GTPVTVSA

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 5 / Number of copies: 2 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Macromolecule #6: (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: 6 / 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 #7: GADOLINIUM ATOM

MacromoleculeName: GADOLINIUM ATOM / type: ligand / ID: 7 / Number of copies: 2 / Formula: GD
Molecular weightTheoretical: 157.25 Da
Chemical component information

ChemComp-GD:
GADOLINIUM ATOM

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.0.1) / Number images used: 132288
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.0.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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