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- EMDB-44850: Cryo-EM structure of the S. cerevisiae lipid flippase Neo1 bound ... -

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Basic information

Entry
Database: EMDB / ID: EMD-44850
TitleCryo-EM structure of the S. cerevisiae lipid flippase Neo1 bound with PI4P in the E2P state
Map data
Sample
  • Complex: Neo1 bound with PI4P
    • Protein or peptide: Phospholipid-transporting ATPase NEO1
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: (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]
Keywordslipid flippase / P4-ATPase / PI4P / phosphoinositide / neomycin resistance / LIPID TRANSPORT
Function / homology
Function and homology information


lysophosphatidylserine flippase activity / trans-Golgi network membrane organization / Ion transport by P-type ATPases / phosphatidylserine flippase activity / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity / vacuole organization / phosphatidylethanolamine flippase activity / 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 / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity / vacuole organization / phosphatidylethanolamine flippase activity / 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 / Cation transport ATPase (P-type) / E1-E2 ATPase / 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 / Cation transport ATPase (P-type) / E1-E2 ATPase / 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
Phospholipid-transporting ATPase NEO1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsDuan HD / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
CitationJournal: 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, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44850.png

Downloads & links

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Map

FileDownload / File: emd_44850.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 238.464 Å		0.83 Å/pix.
x 288 pix.
= 238.464 Å		0.83 Å/pix.
x 288 pix.
= 238.464 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.6925236 - 1.0101846
Average (Standard dev.)0.0004431291 (±0.022009399)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 238.464 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44850_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_44850_half_map_2.map
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Sample components

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Entire : Neo1 bound with PI4P

EntireName: Neo1 bound with PI4P
Components
  • Complex: Neo1 bound with PI4P
    • Protein or peptide: Phospholipid-transporting ATPase NEO1
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: (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]

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Supramolecule #1: Neo1 bound with PI4P

SupramoleculeName: Neo1 bound with PI4P / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Phospholipid-transporting ATPase NEO1

MacromoleculeName: Phospholipid-transporting ATPase NEO1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 131.360469 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MDYKDDDDKP NPPSFKSHKQ NLFNSNNNQH ANSVDSFDLH LDDSFDAALD SLQINNNPEP LSKHNTVGDR ESFEMRTVDD LDNFSNHSS DSHRKSSNTD THPLMYDNRL SQDDNFKFTN IASSPPSSSN NIFSKALSYL KVSNTKNWSK FGSPIELSDQ H IEREIHPD ...String:
MDYKDDDDKP NPPSFKSHKQ NLFNSNNNQH ANSVDSFDLH LDDSFDAALD SLQINNNPEP LSKHNTVGDR ESFEMRTVDD LDNFSNHSS DSHRKSSNTD THPLMYDNRL SQDDNFKFTN IASSPPSSSN NIFSKALSYL KVSNTKNWSK FGSPIELSDQ H IEREIHPD TTPVYDRNRY VSNELSNAKY NAVTFVPTLL YEQFKFFYNL YFLVVALSQA VPALRIGYLS SYIVPLAFVL TV TMAKEAI DDIQRRRRDR ESNNELYHVI TRNRSIPSKD LKVGDLIKVH KGDRIPADLV LLQSSEPSGE SFIKTDQLDG ETD WKLRVA CPLTQNLSEN DLINRISITA SAPEKSIHKF LGKVTYKDST SNPLSVDNTL WANTVLASSG FCIACVVYTG RDTR QAMNT TTAKVKTGLL ELEINSISKI LCACVFALSI LLVAFAGFHN DDWYIDILRY LILFSTIIPV SLRVNLDLAK SVYAH QIEH DKTIPETIVR TSTIPEDLGR IEYLLSDKTG TLTQNDMQLK KIHLGTVSYT SETLDIVSDY VQSLVSSKND SLNNSK VAL STTRKDMSFR VRDMILTLAI CHNVTPTFED DELTYQAASP DEIAIVKFTE SVGLSLFKRD RHSISLLHEH SGKTLNY EI LQVFPFNSDS KRMGIIVRDE QLDEYWFMQK GADTVMSKIV ESNDWLEEET GNMAREGLRT LVIGRKKLNK KIYEQFQK E YNDASLSMLN RDQQMSQVIT KYLEHDLELL GLTGVEDKLQ KDVKSSIELL RNAGIKIWML TGDKVETARC VSISAKLIS RGQYVHTITK VTRPEGAFNQ LEYLKINRNA CLLIDGESLG MFLKHYEQEF FDVVVHLPTV IACRCTPQQK ADVALVIRKM TGKRVCCIG DGGNDVSMIQ CADVGVGIVG KEGKQASLAA DFSITQFCHL TELLLWHGRN SYKRSAKLAQ FVMHRGLIIA I CQAVYSIC SLFEPIALYQ GWLMVGYATC YTMAPVFSLT LDHDIEESLT KIYPELYKEL TEGKSLSYKT FFVWVLLSLF QG SVIQLFS QAFTSLLDTD FTRMVAISFT ALVVNELIMV ALEIYTWNKT MLVTEIATLL FYIVSVPFLG DYFDLGYMTT VNY YAGLLV ILLISIFPVW TAKAIYRRLH PPSYAKVQEF ATP

UniProtKB: Phospholipid-transporting ATPase NEO1

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Macromolecule #2: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #3: (2R)-3-{[(S)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-...

MacromoleculeName: (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]
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1ARJ
Molecular weightTheoretical: 943.086 Da

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1082029
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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