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- PDB-9bd6: PaMsbA in an occluded, outward conformation -

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Basic information

Entry
Database: PDB / ID: 9bd6
TitlePaMsbA in an occluded, outward conformation
ComponentsATP-dependent lipid A-core flippase
KeywordsTRANSLOCASE / MsbA / zinc / membrane protein
Function / homology
Function and homology information


ATP transmembrane transporter activity / ABC-type lipid A-core oligosaccharide transporter / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / lipid A biosynthetic process / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / transmembrane transport / ATP hydrolysis activity / ATP 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
ADP METAVANADATE / ATP-dependent lipid A-core flippase
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.58 Å
AuthorsBahramimoghaddam, H. / Laganowsky, A.
Funding support United States, 3items
OrganizationGrant numberCountry
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)GM139876 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM1454316 United States
CitationJournal: J Am Chem Soc / Year: 2025
Title: Molecular Basis for the Activation of MsbA by Divalent Metals.
Authors: Jixing Lyu / Hanieh Bahramimoghaddam / Tianqi Zhang / Elena Scott / Sangho D Yun / Gaya P Yadav / Minglei Zhao / David Russell / Arthur Laganowsky /
Abstract: Proteins involved in the biogenesis of lipopolysaccharide (LPS), a lipid exclusive to Gram-negative bacteria, are promising candidates for drug discovery. Specifically, the ABC transporter MsbA plays ...Proteins involved in the biogenesis of lipopolysaccharide (LPS), a lipid exclusive to Gram-negative bacteria, are promising candidates for drug discovery. Specifically, the ABC transporter MsbA plays a crucial role in translocating an LPS precursor from the cytoplasmic to the periplasmic facing leaflet of the inner membrane, and small molecules that inhibit its function exhibit bactericidal activity. Here, we use native mass spectrometry (MS) to determine lipid binding affinities of MsbA from (PaMsbA), a Gram-negative bacteria associated with hospital-acquired infections, in different conformations. Unlike the transporter from , we show that the ATPase activity of PaMsbA is stimulated by Zn, Ni, and Mn and successfully trapping the protein with vanadate requires one of these metal ions. We also present cryogenic-electron microscopy structures of PaMsbA in occluded and open outward-facing conformations determined to resolutions of 2.58 and 2.44 Å, respectively. The structures reveal a triad of histidine residues, and mutation of these residues abolishes Zn binding and stimulation of PaMsbA activity by metal ions. Together, our studies provide insight into the structure of PaMsbA and its lipid binding preferences and reveal that a subset of divalent metals stimulates its ATPase activity.
History
DepositionApr 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 17, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATP-dependent lipid A-core flippase
B: ATP-dependent lipid A-core flippase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,74010
Polymers133,2942
Non-polymers1,4478
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-dependent lipid A-core flippase / Lipid A export ATP-binding/permease protein MsbA


Mass: 66646.852 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: msbA, PA4997 / Plasmid: pCDF / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 A1
References: UniProt: Q9HUG8, ABC-type lipid A-core oligosaccharide transporter
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-AD9 / ADP METAVANADATE


Mass: 527.149 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P2V / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: MsbA from Pseudomonas aeruginosa / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.23 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: BL21 A1 / Plasmid: pCDF
Buffer solutionpH: 7.4 / Details: 100mM NaCl, 20mM HEPES, 0.02% DDM
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 50.73 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3138

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
4cryoSPARC4.4CTF correction
7UCSF Chimeramodel fitting
9Cootmodel refinement
10PHENIXmodel refinement
11cryoSPARC4.4initial Euler assignment
12cryoSPARC4.4final Euler assignment
13cryoSPARC4.4classification
14cryoSPARC4.43D reconstruction
Image processingDetails: Image processing and reconstruction was performed using cryoSPARC.
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1915839
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 232095 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial local fitting was done using Chimera, manual model building in Coot, and one round of real space refinement in Phenix
Atomic model buildingPDB-ID: 4WFF

4wff


Pdb chain-ID: A / Accession code: 4WFF / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.58 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039178
ELECTRON MICROSCOPYf_angle_d0.61812444
ELECTRON MICROSCOPYf_dihedral_angle_d4.6741258
ELECTRON MICROSCOPYf_chiral_restr0.0411464
ELECTRON MICROSCOPYf_plane_restr0.0161572

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