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- PDB-8tsp: Open, inward-facing MsbA structure (OIF1) -

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Basic information

Entry
Database: PDB / ID: 8tsp
TitleOpen, inward-facing MsbA structure (OIF1)
ComponentsATP-binding transport protein MsbA
KeywordsTRANSPORT PROTEIN / ABC transporter
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / ABC-type oligopeptide transporter activity / ATPase-coupled lipid transmembrane transporter activity / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
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 ...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-binding transport protein MsbA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsYang, B. / Zhang, T. / Lyu, J. / Laganowsky, A.D. / Zhao, M.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121751 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM139876 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM138863 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM145416 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143052 United States
CitationJournal: Nat Commun / Year: 2024
Title: Native mass spectrometry and structural studies reveal modulation of MsbA-nucleotide interactions by lipids.
Authors: Tianqi Zhang / Jixing Lyu / Bowei Yang / Sangho D Yun / Elena Scott / Minglei Zhao / Arthur Laganowsky /
Abstract: The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the ...The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.
History
DepositionAug 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-binding transport protein MsbA
B: ATP-binding transport protein MsbA


Theoretical massNumber of molelcules
Total (without water)129,0872
Polymers129,0872
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ATP-binding transport protein MsbA / ATP-binding transport protein multicopy suppressor of htrB / Lipid A ABC exporter / fused ATPase ...ATP-binding transport protein multicopy suppressor of htrB / Lipid A ABC exporter / fused ATPase and inner membrane subunits MsbA / Lipid A ABC transporter ATP-binding protein/permease MsbA / Lipid A export ATP-binding/permease MsbA / Lipid A export permease MsbA / Lipid ABC transporter permease/ATP-binding protein / Lipid ABC transporter permease/ATPase / Lipid transporter ATP-binding/permease / Vga(B)_1_U82085


Mass: 64543.473 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
References: UniProt: C3TGA2, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
Has protein modificationN

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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 incubated with ATP for 6 hours / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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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: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111791 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059008
ELECTRON MICROSCOPYf_angle_d0.75412178
ELECTRON MICROSCOPYf_dihedral_angle_d4.2431244
ELECTRON MICROSCOPYf_chiral_restr0.0411454
ELECTRON MICROSCOPYf_plane_restr0.011540

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