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- PDB-9vdm: Cryo-EM structure of human ATP9A (AMPPCP) E2P state open form -

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Entry
Database: PDB / ID: 9vdm
TitleCryo-EM structure of human ATP9A (AMPPCP) E2P state open form
ComponentsProbable phospholipid-transporting ATPase IIA
KeywordsTRANSPORT PROTEIN / P-type ATPase / flippase / human
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
CHOLESTEROL / Chem-P5S / Probable phospholipid-transporting ATPase IIA
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsAbe, K.
Funding support Japan, 1items
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 homolog, 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 homolog, 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 2.2 Å, 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 headgroups 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 with 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, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Probable phospholipid-transporting ATPase IIA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)116,1754
Polymers114,9721
Non-polymers1,2033
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 Probable phospholipid-transporting ATPase IIA / ATPase class II type 9A


Mass: 114971.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATP9A, ATPIIA, KIAA0611 / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: O75110, P-type phospholipid transporter
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-P5S / O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine / phosphatidyl serine


Mass: 792.075 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO10P / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
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: human ATP9A / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 6.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 10000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59377 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 87.99 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00258141
ELECTRON MICROSCOPYf_angle_d0.55411041
ELECTRON MICROSCOPYf_chiral_restr0.04161283
ELECTRON MICROSCOPYf_plane_restr0.0031379
ELECTRON MICROSCOPYf_dihedral_angle_d8.19781121

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