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- PDB-9bs1: Cryo-EM structure of the S. cerevisiae lipid flippase Neo1 bound ... -

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Basic information

Entry
Database: PDB / ID: 9bs1
TitleCryo-EM structure of the S. cerevisiae lipid flippase Neo1 bound with PI4P in the E2P state
ComponentsPhospholipid-transporting ATPase NEO1
KeywordsLIPID TRANSPORT / lipid flippase / P4-ATPase / PI4P / phosphoinositide / neomycin resistance
Function / homology
Function and homology information


lysophosphatidylserine flippase activity / trans-Golgi network membrane organization / Ion transport by P-type ATPases / phosphatidylserine flippase activity / phosphatidylserine floppase activity / ATPase-coupled intramembrane lipid transporter activity / phosphatidylethanolamine flippase activity / vacuole organization / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / P-type phospholipid transporter ...lysophosphatidylserine flippase activity / trans-Golgi network membrane organization / Ion transport by P-type ATPases / phosphatidylserine flippase activity / phosphatidylserine floppase activity / ATPase-coupled intramembrane lipid transporter activity / phosphatidylethanolamine flippase activity / vacuole organization / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / P-type phospholipid transporter / phospholipid translocation / trans-Golgi network / endocytosis / late endosome / protein transport / endosome / endosome membrane / Golgi membrane / magnesium ion binding / Golgi apparatus / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / P-type ATPase, cytoplasmic domain N / P-type ATPase actuator domain / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N ...P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / P-type ATPase, cytoplasmic domain N / P-type ATPase actuator domain / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
: / BERYLLIUM TRIFLUORIDE ION / Phospholipid-transporting ATPase NEO1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsDuan, H.D. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
Citation
Journal: Nat Cell Biol / Year: 2025
Title: P4-ATPases control phosphoinositide membrane asymmetry and neomycin resistance.
Authors: Bhawik K Jain / H Diessel Duan / Christina Valentine / Ariana Samiha / Huilin Li / Todd R Graham /
Abstract: The aminoglycoside antibiotic neomycin has robust antibacterial properties, yet its clinical utility is curtailed by its nephrotoxicity and ototoxicity. The mechanism by which the polycationic ...The aminoglycoside antibiotic neomycin has robust antibacterial properties, yet its clinical utility is curtailed by its nephrotoxicity and ototoxicity. The mechanism by which the polycationic neomycin enters specific eukaryotic cell types remains poorly understood. In budding yeast, NEO1 is required for neomycin resistance and encodes a phospholipid flippase that establishes membrane asymmetry. Here we show that mutations altering Neo1 substrate recognition cause neomycin hypersensitivity by exposing phosphatidylinositol-4-phosphate (PI4P) in the plasma membrane extracellular leaflet. Cryogenic electron microscopy reveals PI4P binding to Neo1 within the substrate translocation pathway. PI4P enters the lumen of the endoplasmic reticulum and is flipped by Neo1 at the Golgi to prevent PI4P secretion to the cell surface. Deficiency of the orthologous ATP9A in human cells also causes exposure of PI4P and neomycin sensitivity. These findings unveil conserved mechanisms of aminoglycoside sensitivity and phosphoinositide homoeostasis, with important implications for signalling by extracellular phosphoinositides.
#1: Journal: bioRxiv / Year: 2025
Title: P4-ATPase control over phosphoinositide membrane asymmetry and neomycin resistance.
Authors: Bhawik K Jain / H Diessel Duan / Christina Valentine / Ariana Samiha / Huilin Li / Todd R Graham /
Abstract: Neomycin, an aminoglycoside antibiotic, has robust antibacterial properties, yet its clinical utility is curtailed by its nephrotoxicity and ototoxicity. The mechanism by which the polycationic ...Neomycin, an aminoglycoside antibiotic, has robust antibacterial properties, yet its clinical utility is curtailed by its nephrotoxicity and ototoxicity. The mechanism by which the polycationic neomycin enters specific eukaryotic cell types remains poorly understood. In budding yeast, is required for neomycin resistance and encodes a phospholipid flippase that establishes membrane asymmetry. Here, we show that mutations altering Neo1 substrate recognition cause neomycin hypersensitivity by exposing phosphatidylinositol-4-phosphate (PI4P) in the plasma membrane extracellular leaflet. Human cells also expose extracellular PI4P upon knockdown of ATP9A, a Neo1 ortholog and ATP9A expression level correlates to neomycin sensitivity. In yeast, the extracellular PI4P is initially produced in the cytosolic leaflet of the plasma membrane and then delivered by Osh6-dependent nonvesicular transport to the endoplasmic reticulum (ER). Here, a portion of PI4P escapes degradation by the Sac1 phosphatase by entering the ER lumenal leaflet. COPII vesicles transport lumenal PI4P to the Golgi where Neo1 flips this substrate back to the cytosolic leaflet. Cryo-EM reveals that PI4P binds Neo1 within the substrate translocation pathway. Loss of Neo1 activity in the Golgi allows secretion of extracellular PI4P, which serves as a neomycin receptor and facilitates its endocytic uptake. These findings unveil novel mechanisms of aminoglycoside sensitivity and phosphoinositide homeostasis, with important implications for signaling by extracellular phosphoinositides.
History
DepositionMay 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 2.0Jul 23, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Structure summary
Category: atom_site / citation ...atom_site / citation / citation_author / em_admin / pdbx_contact_author / pdbx_unobs_or_zero_occ_atoms
Item: _em_admin.last_update / Description: Model completeness
Details: The side chain of Asn168 has been corrected . Previously the side chain was shown incomplete, i.e., missing 3 non-hydrogen atoms.
Provider: author / Type: Coordinate replacement
Revision 2.1Jul 30, 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: Phospholipid-transporting ATPase NEO1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,3703
Polymers131,3601
Non-polymers1,0092
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 Phospholipid-transporting ATPase NEO1


Mass: 131360.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NEO1, YIL048W / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P40527, P-type phospholipid transporter
#2: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3
#3: Chemical ChemComp-A1ARJ / (2R)-3-{[(S)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl di[(9Z)-octadec-9-enoate]


Mass: 943.086 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C45H84O16P2 / 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: Neo1 bound with PI4P / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1082029 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027837
ELECTRON MICROSCOPYf_angle_d0.53110627
ELECTRON MICROSCOPYf_dihedral_angle_d5.161048
ELECTRON MICROSCOPYf_chiral_restr0.0381254
ELECTRON MICROSCOPYf_plane_restr0.0031326

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