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- PDB-7ph2: Nanodisc reconstituted MsbA in complex with nanobodies, spin-labe... -

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Basic information

Entry
Database: PDB / ID: 7ph2
TitleNanodisc reconstituted MsbA in complex with nanobodies, spin-labeled at position A60C
Components
  • ATP-dependent lipid A-core flippase
  • Nb_MsbA#1
KeywordsMEMBRANE PROTEIN / ABC transporter / nanobody / Gd-DOTA
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 transmembrane region fold / ABC transporter type 1, transmembrane domain / 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 ...Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter transmembrane region fold / ABC transporter type 1, transmembrane domain / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Immunoglobulins / Up-down Bundle / Immunoglobulin-like / Sandwich / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chem-88T / Chem-EIW / GADOLINIUM ATOM / ATP-dependent lipid A-core flippase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsJanuliene, D. / Parey, K. / Galazzo, L. / Meier, G. / Vecchis, D. / Striednig, B. / Hilbi, H. / Schaefer, L.V. / Kuprov, I. / Bordignon, E. ...Januliene, D. / Parey, K. / Galazzo, L. / Meier, G. / Vecchis, D. / Striednig, B. / Hilbi, H. / Schaefer, L.V. / Kuprov, I. / Bordignon, E. / Seeger, M.A. / Moeller, A.
Funding support Germany, 3items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 24, 2022Provider: repository / Type: Initial release
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Revision 1.2Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-dependent lipid A-core flippase
B: ATP-dependent lipid A-core flippase
C: Nb_MsbA#1
D: Nb_MsbA#1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,8329
Polymers156,2614
Non-polymers3,5705
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15630 Å2
ΔGint-109 kcal/mol
Surface area66460 Å2

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Components

#1: Protein ATP-dependent lipid A-core flippase / Lipid A export ATP-binding/permease protein MsbA / Lipid flippase


Mass: 65760.852 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: msbA, b0914, JW0897 / Production host: Escherichia coli (E. coli)
References: UniProt: P60752, ABC-type lipid A-core oligosaccharide transporter
#2: Antibody Nb_MsbA#1


Mass: 12369.879 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Nanobody is labeled with GMO-GD at Cys60 / Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
#3: Chemical 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


Mass: 2206.503 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C102H186N2O44P2
#4: Chemical 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


Mass: 524.524 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H32N6O9
#5: Chemical ChemComp-GD / GADOLINIUM ATOM


Mass: 157.250 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Gd
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E. coli MsbA in complex with nanobody Nb_MsbA#1, labeled with Gd-DOTA at Cys60
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
8PHENIXmodel refinement
10RELION3.1initial Euler assignment
13cryoSPARCv3.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142832 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610846
ELECTRON MICROSCOPYf_angle_d0.77114645
ELECTRON MICROSCOPYf_dihedral_angle_d17.9571560
ELECTRON MICROSCOPYf_chiral_restr0.0451727
ELECTRON MICROSCOPYf_plane_restr0.0051844

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