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- PDB-6k7h: Cryo-EM structure of the human P4-type flippase ATP8A1-CDC50 (E1 ... -

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Basic information

Entry
Database: PDB / ID: 6k7h
TitleCryo-EM structure of the human P4-type flippase ATP8A1-CDC50 (E1 state class2)
Components
  • Cell cycle control protein 50A
  • Phospholipid-transporting ATPase
KeywordsMEMBRANE PROTEIN / flippase
Function / homology
Function and homology information


aminophospholipid flippase activity / flippase activity / ATPase-coupled intramembrane lipid transporter activity / positive regulation of phospholipid translocation / aminophospholipid transport / positive regulation of protein exit from endoplasmic reticulum / protein localization to endosome / chromaffin granule membrane / P-type phospholipid transporter / ATPase-coupled cation transmembrane transporter activity ...aminophospholipid flippase activity / flippase activity / ATPase-coupled intramembrane lipid transporter activity / positive regulation of phospholipid translocation / aminophospholipid transport / positive regulation of protein exit from endoplasmic reticulum / protein localization to endosome / chromaffin granule membrane / P-type phospholipid transporter / ATPase-coupled cation transmembrane transporter activity / drug transmembrane transport / phospholipid transport / phospholipid translocation / azurophil granule membrane / transmembrane transporter activity / lipid transport / transport vesicle membrane / specific granule membrane / ion transmembrane transport / positive regulation of neuron projection development / learning / trans-Golgi network / transmembrane transport / apical plasma membrane / ATPase activity / positive regulation of cell migration / intracellular membrane-bounded organelle / neutrophil degranulation / Golgi apparatus / endoplasmic reticulum / magnesium ion binding / extracellular exosome / membrane / integral component of membrane / ATP binding / plasma membrane
LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, cytoplasmic domain N / P-type ATPase / CDC50/LEM3 family / P-type ATPase, subfamily IV / P-type ATPase, A domain superfamily / P-type ATPase, phosphorylation site / HAD superfamily / P-type ATPase, transmembrane domain superfamily / Cell cycle control protein 50A ...LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, cytoplasmic domain N / P-type ATPase / CDC50/LEM3 family / P-type ATPase, subfamily IV / P-type ATPase, A domain superfamily / P-type ATPase, phosphorylation site / HAD superfamily / P-type ATPase, transmembrane domain superfamily / Cell cycle control protein 50A / P-type ATPase, C-terminal / P-type ATPase, N-terminal / HAD-like superfamily / E1-E2 ATPase / Cation transport ATPase (P-type) / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal
Phospholipid-transporting ATPase / Cell cycle control protein 50A / Phospholipid-transporting ATPase IA
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsHiraizumi, M. / Yamashita, K. / Nishizawa, T. / Nureki, O.
CitationJournal: Science / Year: 2019
Title: Cryo-EM structures capture the transport cycle of the P4-ATPase flippase.
Authors: Masahiro Hiraizumi / Keitaro Yamashita / Tomohiro Nishizawa / Osamu Nureki /
Abstract: In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is ...In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Here, we report the cryo-electron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 7, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 6, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB

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Assembly

Deposited unit
A: Phospholipid-transporting ATPase
C: Cell cycle control protein 50A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,8178
Polymers171,2652
Non-polymers1,5526
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9720 Å2
ΔGint-34 kcal/mol
Surface area56500 Å2

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Components

#1: Protein/peptide Phospholipid-transporting ATPase


Mass: 130537.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3.4 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: Q59EX4, UniProt: Q9Y2Q0*PLUS, P-type phospholipid transporter
#2: Protein/peptide Cell cycle control protein 50A


Mass: 40727.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC50A / Plasmid: pcDNA3.4 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9NV96
#3: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MAN / ALPHA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: ATP8A1-CDC50a / Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F / Plasmid: pcDNA3.4
Buffer solutionpH: 7.5
SpecimenConc.: 4 mg/ml / Embedding 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 424465 / Symmetry type: POINT
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.010810836
f_angle_d1.093814721
f_chiral_restr0.06441682
f_plane_restr0.00811842
f_dihedral_angle_d5.27266446

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