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- PDB-7vsg: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted... -

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Basic information

Entry
Database: PDB / ID: 7vsg
TitleCryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in PtdSer-occluded E2-Pi state.
Components
  • Cell cycle control protein 50A
  • Phospholipid-transporting ATPase IG
KeywordsTRANSPORT PROTEIN / flippase / P4-ATPase / membrane protein / phospholipid transporter
Function / homology
Function and homology information


positive regulation of phospholipid translocation / aminophospholipid flippase activity / aminophospholipid transport / phosphatidylserine flippase activity / protein localization to endosome / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity / positive regulation of protein exit from endoplasmic reticulum / xenobiotic transmembrane transport ...positive regulation of phospholipid translocation / aminophospholipid flippase activity / aminophospholipid transport / phosphatidylserine flippase activity / protein localization to endosome / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity / positive regulation of protein exit from endoplasmic reticulum / xenobiotic transmembrane transport / phosphatidylethanolamine flippase activity / P-type phospholipid transporter / phospholipid translocation / transport vesicle membrane / azurophil granule membrane / Ion transport by P-type ATPases / specific granule membrane / monoatomic ion transmembrane transport / recycling endosome / positive regulation of neuron projection development / recycling endosome membrane / late endosome membrane / early endosome membrane / apical plasma membrane / lysosomal membrane / Neutrophil degranulation / endoplasmic reticulum membrane / structural molecule activity / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / 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 ...CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / 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
Chem-17F / Phospholipid-transporting ATPase IG / Cell cycle control protein 50A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsNakanishii, H. / Abe, K.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21K06064 Japan
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: J Biol Chem / Year: 2022
Title: Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2.
Authors: Hanayo Nakanishi / Kenichi Hayashida / Tomohiro Nishizawa / Atsunori Oshima / Kazuhiro Abe /
Abstract: ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C ...ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-β-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.
History
DepositionOct 26, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 29, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)167,0136
Polymers165,1962
Non-polymers1,8174
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9440 Å2
ΔGint-30 kcal/mol
Surface area48420 Å2

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Components

#1: Protein Phospholipid-transporting ATPase IG / ATPase IQ / ATPase class VI type 11C / P4-ATPase flippase complex alpha subunit ATP11C


Mass: 124468.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATP11C, ATPIG, ATPIQ / Production host: Mammalia (mammals)
References: UniProt: Q8NB49, P-type phospholipid transporter
#2: Protein 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 / Production host: Mammalia (mammals) / References: UniProt: Q9NV96
#3: Polysaccharide alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{}}}LINUCSPDB-CARE
#4: Chemical ChemComp-17F / O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy)phosphoryl]-L-serine / 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine


Mass: 788.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H78NO10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
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: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in PtdSer-occluded E2-Pi state.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalia (mammals)
Buffer solutionpH: 6.5
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143384 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029553
ELECTRON MICROSCOPYf_angle_d0.63512952
ELECTRON MICROSCOPYf_dihedral_angle_d8.0891320
ELECTRON MICROSCOPYf_chiral_restr0.0541468
ELECTRON MICROSCOPYf_plane_restr0.0041610

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