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- EMDB-64988: Cryo-EM structure of human ATP9A (AMPPCP) E2P state open form -

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Basic information

Entry
Database: EMDB / ID: EMD-64988
TitleCryo-EM structure of human ATP9A (AMPPCP) E2P state open form
Map data
Sample
  • Complex: human ATP9A
    • Protein or peptide: Probable phospholipid-transporting ATPase IIA
  • Ligand: MAGNESIUM ION
  • Ligand: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine
  • Ligand: CHOLESTEROL
KeywordsP-type ATPase / flippase / human / TRANSPORT PROTEIN
Function / homology
Function and homology information


regulation of endocytic recycling / negative regulation of exosomal secretion / ATPase-coupled intramembrane lipid transporter activity / regulation of retrograde transport, endosome to Golgi / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / P-type phospholipid transporter / phospholipid translocation / Ion transport by P-type ATPases / neuron projection morphogenesis / trans-Golgi network membrane ...regulation of endocytic recycling / negative regulation of exosomal secretion / ATPase-coupled intramembrane lipid transporter activity / regulation of retrograde transport, endosome to Golgi / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / P-type phospholipid transporter / phospholipid translocation / Ion transport by P-type ATPases / neuron projection morphogenesis / trans-Golgi network membrane / trans-Golgi network / recycling endosome / endocytosis / recycling endosome membrane / late endosome membrane / late endosome / protease binding / early endosome membrane / early endosome / endosome / perinuclear region of cytoplasm / magnesium ion binding / 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 / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Probable phospholipid-transporting ATPase IIA
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsAbe K / Blanco G
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)24K01975 Japan
CitationJournal: J Biol Chem / Year: 2025
Title: A unique gating mechanism revealed by the cryo-EM structure of monomeric ATP9A flippase.
Authors: Kazuhiro Abe / Parthiban Marimuthu / Yuheng Qian / Chai C Gopalasingam / Christoph Gerle / Hideki Shigematsu / Kotaro Tanaka / Himanshu Khandelia /
Abstract: Among mammalian P4-ATPase flippases, only ATP9A and ATP9B do not require the auxiliary subunit CDC50 protein. Whilst its yeast homologue, Neo1, is essential for cell survival, little is known about ...Among mammalian P4-ATPase flippases, only ATP9A and ATP9B do not require the auxiliary subunit CDC50 protein. Whilst its yeast homologue, Neo1, is essential for cell survival, little is known about mammalian ATP9A. We present cryo-EM structures of human monomeric ATP9A at a resolution reaching to 2.2Å, in the outward-facing E2P state. Two distinguishable conformations were obtained from a single sample, one with its outward gate open, and the other in its closed form. Unlike canonical gating observed for most P-type ATPases, which is driven by the movement of transmembrane (TM) helices 1 and 2 linked to the A domain, outward gating in ATP9A is achieved by the movement of TM6-10 helices, likely initiated by the unwinding of TM6. As a result, the volume of the phospholipid binding cavity in the open state surpasses that of other flippases, which could allow binding of phospholipids with larger hydrophilic head groups than that of phosphatidylserine. ATP9A shows an ATPase activity that is significantly increased by the addition of phospholipids that retain the overall negative charge, including phosphatidylserine, phosphatidylinositol and its phosphorylated species, compared to other electroneutral phospholipids. The observation of spontaneous binding of phosphorylated species of phosphatidylinositol in molecular simulation reinforces this fact. Our data provide mechanistic rationales for ATP9A gating, achieved by the rearrangement of the second half of the TM helices. Since TM4 - TM10 is anchored by the CDC50 protein subunit in other flippases, the here observed outward gating mechanism is unique to P4B-type flippases which function as a monomer.
History
DepositionJun 9, 2025-
Header (metadata) releaseSep 3, 2025-
Map releaseSep 3, 2025-
UpdateSep 10, 2025-
Current statusSep 10, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64988.png

Downloads & links

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Map

FileDownload / File: emd_64988.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 450 pix.
= 338.4 Å		0.75 Å/pix.
x 450 pix.
= 338.4 Å		0.75 Å/pix.
x 450 pix.
= 338.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.752 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.361
Minimum - Maximum-1.4103775 - 2.346552
Average (Standard dev.)-0.0013784817 (±0.040373735)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 338.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_64988_msk_1.map
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Half map: #1

Fileemd_64988_half_map_1.map
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Half map: #2

Fileemd_64988_half_map_2.map
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Sample components

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Entire : human ATP9A

EntireName: human ATP9A
Components
  • Complex: human ATP9A
    • Protein or peptide: Probable phospholipid-transporting ATPase IIA
  • Ligand: MAGNESIUM ION
  • Ligand: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine
  • Ligand: CHOLESTEROL

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Supramolecule #1: human ATP9A

SupramoleculeName: human ATP9A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Probable phospholipid-transporting ATPase IIA

MacromoleculeName: Probable phospholipid-transporting ATPase IIA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 114.971773 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GGEARPRTVW LGHPEKRDQR YPRNVINNQK YNFFTFLPGV LFNQFKYFFN LYFLLLACSQ FVPEMRLGAL YTYWVPLGFV LAVTVIREA VEEIRCYVRD KEVNSQVYSR LTARGTVKVK SSNIQVGDLI IVEKNQRVPA DMIFLRTSEK NGSCFLRTDQ L DGETDWKL ...String:
GGEARPRTVW LGHPEKRDQR YPRNVINNQK YNFFTFLPGV LFNQFKYFFN LYFLLLACSQ FVPEMRLGAL YTYWVPLGFV LAVTVIREA VEEIRCYVRD KEVNSQVYSR LTARGTVKVK SSNIQVGDLI IVEKNQRVPA DMIFLRTSEK NGSCFLRTDQ L DGETDWKL RLPVACTQRL PTAADLLQIR SYVYAEEPNI DIHNFVGTFT REDSDPPISE SLSIENTLWA GTVVASGTVV GV VLYTGRE LRSVMNTSNP RSKIGLFDLE VNCLTKILFG ALVVVSLVMV ALQHFAGRWY LQIIRFLLLF SNIIPISLRV NLD MGKIVY SWVIRRDSKI PGTVVRSSTI PEQLGRISYL LTDKTGTLTQ NEMIFKRLHL GTVAYGLDSM DEVQSHIFSI YTQQ SQDPP AQKGPTLTTK VRRTMSSRVH EAVKAIALCH NVTPVYESNG VTDQAEAEKQ YEDSCRVYQA SSPDEVALVQ WTESV GLTL VGRDQSSMQL RTPGDQILNF TILQIFPFTY ESKRMGIIVR DESTGEITFY MKGADVVMAG IVQYNDWLEE ECGNMA REG LRVLVVAKKS LAEEQYQDFE ARYVQAKLSV HDRSLKVATV IESLEMEMEL LCLTGVEDQL QADVRPTLET LRNAGIK VW MLTGDKLETA TCTAKNAHLV TRNQDIHVFR LVTNRGEAHL ELNAFRRKHD CALVISGDSL EVCLKYYEYE FMELACQC P AVVCCRCAPT QKAQIVRLLQ ERTGKLTCAV GDGGNDVSMI QESDCGVGVE GKEGKQASLA ADFSITQFKH LGRLLMVHG RNSYKRSAAL SQFVIHRSLC ISTMQAVFSS VFYFASVPLY QGFLIIGYST IYTMFPVFSL VLDKDVKSEV AMLYPELYKD LLKGRPLSY KTFLIWVLIS IYQGSTIMYG ALLLFESEFV HIVAISFTSL ILTELLMVAL TIQTWHWLMT VAELLSLACY I ASLVFLHE FIDVYFIATL SFLWKVSVIT LVSCLPLYVL KYLRRRFSPP SYSKLTS

UniProtKB: Probable phospholipid-transporting ATPase IIA

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phospho...

MacromoleculeName: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine
type: ligand / ID: 3 / Number of copies: 1 / Formula: P5S
Molecular weightTheoretical: 792.075 Da
Chemical component information

ChemComp-P5S:
O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine

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Macromolecule #4: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 4 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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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: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 10.0 µm / Nominal defocus min: 1.0 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7bsu

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 59377
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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