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- EMDB-44444: PaMsbA in an occluded, outward conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-44444
TitlePaMsbA in an occluded, outward conformation
Map data
Sample
  • Complex: MsbA from Pseudomonas aeruginosa
    • Protein or peptide: ATP-dependent lipid A-core flippase
  • Ligand: ZINC ION
  • Ligand: ADP METAVANADATE
KeywordsMsbA / zinc / membrane protein / TRANSLOCASE
Function / homology
Function and homology information


ATP transmembrane transporter activity / ABC-type lipid A-core oligosaccharide transporter / ATPase-coupled lipid transmembrane transporter activity / lipid A biosynthetic process / lipopolysaccharide transport / 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
ATP-dependent lipid A-core flippase
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.58 Å
AuthorsBahramimoghaddam H / Laganowsky A
Funding support United States, 3 items
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, 2024-
Header (metadata) releaseSep 3, 2025-
Map releaseSep 3, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44444.png

Downloads & links

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Map

FileDownload / File: emd_44444.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 270 pix.
= 224.64 Å		0.83 Å/pix.
x 270 pix.
= 224.64 Å		0.83 Å/pix.
x 270 pix.
= 224.64 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.0052329786 - 0.25744134
Average (Standard dev.)0.00041139382 (±0.0074074403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions270270270
Spacing270270270
CellA=B=C: 224.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44444_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44444_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : MsbA from Pseudomonas aeruginosa

EntireName: MsbA from Pseudomonas aeruginosa
Components
  • Complex: MsbA from Pseudomonas aeruginosa
    • Protein or peptide: ATP-dependent lipid A-core flippase
  • Ligand: ZINC ION
  • Ligand: ADP METAVANADATE

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Supramolecule #1: MsbA from Pseudomonas aeruginosa

SupramoleculeName: MsbA from Pseudomonas aeruginosa / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 1.23 MDa

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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: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 66.646852 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GSMSDSPQNP GPSSLKIYFR LLGYVKPYIG MFLLSIVGFL IFASTQPMLA GILKYFVDGL SNPDAALFPN VQWPWLRDLH LVYAVPLLI ILIAAWQGLG SFLGNFFLAK VSLGLVHDLR VALFNKLLVL PNRYFDTHSS GHLISRITFN VTMVTGAATD A IKVVIREG ...String:
GSMSDSPQNP GPSSLKIYFR LLGYVKPYIG MFLLSIVGFL IFASTQPMLA GILKYFVDGL SNPDAALFPN VQWPWLRDLH LVYAVPLLI ILIAAWQGLG SFLGNFFLAK VSLGLVHDLR VALFNKLLVL PNRYFDTHSS GHLISRITFN VTMVTGAATD A IKVVIREG LTVVFLFLYL LWMNWKLTLV MLAILPVIAV MVTTASRKFR KQSKKIQVAM GDVTHVASET IQGYRVVRSF GG EAYEEKR FLDASQSNTD KQLRMTKTGA VYTPMLQLVI YVAMAILMFL VLWLRGDASA GDLVAYITAA GLLPKPIRQL SEV SSTVQR GVAGAESIFE QLDEAAEEDQ GTVEKERVSG RLEVRNLSFR YPGTDKQVLD DISFIAEPGQ MIALVGRSGS GKST LANLV PRFYQHNDGK ILLDGVEVED YRLRNLRRHI ALVTQQVTLF NDSVANNIAY GDLAGAPREE IERAAKAANA KEFID NLPQ GFDTEVGENG VLLSGGQRQR LAIARALLKD APLLILDEAT SALDTESERH IQAALDEVMK GRTTLVIAHR LSTIEK ADL ILVMDQGQIV ERGSHAELLA QNGHYARLHA MGLDEQAPAP VG

UniProtKB: ATP-dependent lipid A-core flippase

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: ADP METAVANADATE

MacromoleculeName: ADP METAVANADATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: AD9
Molecular weightTheoretical: 527.149 Da
Chemical component information

ChemComp-AD9:
ADP METAVANADATE

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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

Concentration10 mg/mL
BufferpH: 7.4 / Details: 100mM NaCl, 20mM HEPES, 0.02% DDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 3138 / Average electron dose: 50.73 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImage processing and reconstruction was performed using cryoSPARC.
Particle selectionNumber selected: 1915839
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4) / Type: NONE
Startup modelType of model: OTHER / Details: AlphaFold model AF-Q9HUG8-F1
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 232095
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wff


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting was done using Chimera, manual model building in Coot, and one round of real space refinement in Phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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