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- EMDB-32111: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted... -

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Entry
Database: EMDB / ID: EMD-32111
TitleCryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in E1P state.
Map data
Sample
  • Complex: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in E1P state.
    • Protein or peptide: Phospholipid-transporting ATPase IG
    • Protein or peptide: Cell cycle control protein 50A
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy)phosphoryl]-L-serine
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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
Phospholipid-transporting ATPase IG / Cell cycle control protein 50A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsNakanishii H / Abe K
Funding support Japan, 2 items
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, 2021-
Header (metadata) releaseDec 29, 2021-
Map releaseDec 29, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
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  • Surface level: 0.015
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  • Surface view with fitted model
  • Atomic models: PDB-7vsh
  • Surface level: 0.015
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_32111.png

Downloads & links

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Map

FileDownload / File: emd_32111.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0133 / Movie #1: 0.015
Minimum - Maximum-0.04934602 - 0.0949225
Average (Standard dev.)0.00037079133 (±0.0029516688)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z212.480212.480212.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0490.0950.000

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Supplemental data

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Mask #1

Fileemd_32111_msk_1.map
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Half map: #1

Fileemd_32111_half_map_1.map
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Histogram (log scale)

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Half map: #2

Fileemd_32111_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted...

EntireName: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in E1P state.
Components
  • Complex: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in E1P state.
    • Protein or peptide: Phospholipid-transporting ATPase IG
    • Protein or peptide: Cell cycle control protein 50A
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy)phosphoryl]-L-serine
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted...

SupramoleculeName: Cryo-EM structure of a human ATP11C-CDC50A flippase reconstituted in the Nanodisc in E1P state.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Mammalia (mammals)

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Macromolecule #1: Phospholipid-transporting ATPase IG

MacromoleculeName: Phospholipid-transporting ATPase IG / 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: 124.403617 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: RFCAGEEKRV GTRTVFVGNH PVSETEAYIA QRFCDNRIVS SKYTLWNFLP KNLFEQFRRI ANFYFLIIFL VQVTVDTPTS PVTSGLPLF FVITVTAIKQ GYEDWLRHRA DNEVNKSTVY IIENAKRVRK ESEKIKVGDV VEVQADETFP CDLILLSSCT T DGTCYVTT ...String:
RFCAGEEKRV GTRTVFVGNH PVSETEAYIA QRFCDNRIVS SKYTLWNFLP KNLFEQFRRI ANFYFLIIFL VQVTVDTPTS PVTSGLPLF FVITVTAIKQ GYEDWLRHRA DNEVNKSTVY IIENAKRVRK ESEKIKVGDV VEVQADETFP CDLILLSSCT T DGTCYVTT ASLDGESNCK THYAVRDTIA LCTAESIDTL RAAIECEQPQ PDLYKFVGRI NIYSNSLEAV ARSLGPENLL LK GATLKNT EKIYGVAVYT GMETKMALNY QGKSQKRSAV EKSINAFLIV YLFILLTKAA VCTTLKYVWQ STPYNDEPWY NQK TQKERE TLKVLKMFTD FLSFMVLFNF IIPVSMYVTV EMQKFLGSFF ISWDKDFYDE EINEGALVNT SDLNEELGQV DYVF TDKTG TLTENSMEFI ECCIDGHKYK GVTQEVDGLS QTDGTLTYFD KVDKNREELF LRALCLCHTV EIKTNDAVDG ATESA ELTY ISSSPDEIAL VKGAKRYGFT FLGNRNGYMR VENQRKEIEE YELLHTLNFD AVRRRMSVIV KTQEGDILLF CKGADS AVF PRVQNHEIEL TKVHVERNAM DGYRTLCVAF KEIAPDDYER INRQLIEAKM ALQDREEKME KVFDDIETNM NLIGATA VE DKLQDQAAET IEALHAAGLK VWVLTGDKME TAKSTCYACR LFQTNTELLE LTTKTIEESE RKEDRLHELL IEYRKKLL H EFPKSTRSFK KAWTEHQEYG LIIDGSTLSL ILNSSQDSSS NNYKSIFLQI CMKCTAVLCC RMAPLQKAQI VRMVKNLKG SPITLSIGDG ANDVSMILES HVGIGIKGKE GRQAARNSDY SVPKFKHLKK LLLAHGHLYY VRIAHLVQYF FYKNLCFILP QFLYQFFCG FSQQPLYDAA YLTMYNICFT SLPILAYSLL EQHINIDTLT SDPRLYMKIS GNAMLQLGPF LYWTFLAAFE G TVFFFGTY FLFQTASLEE NGKVYGNWTF GTIVFTVLVF TVTLKLALDT RFWTWINHFV IWGSLAFYVF FSFFWGGIIW PF LKQQRMY FVFAQMLSSV STWLAIILLI FISLFPEILL IVLKNVR

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Macromolecule #2: Cell cycle control protein 50A

MacromoleculeName: Cell cycle control protein 50A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.727527 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: MAMNYNAKDE VDGGPPCAPG GTAKTRRPDN TAFKQQRLPA WQPILTAGTV LPIFFIIGLI FIPIGIGIFV TSNNIREIEI DYTGTEPSS PCNKCLSPDV TPCFCTINFT LEKSFEGNVF MYYGLSNFYQ NHRRYVKSRD DSQLNGDSSA LLNPSKECEP Y RRNEDKPI ...String:
MAMNYNAKDE VDGGPPCAPG GTAKTRRPDN TAFKQQRLPA WQPILTAGTV LPIFFIIGLI FIPIGIGIFV TSNNIREIEI DYTGTEPSS PCNKCLSPDV TPCFCTINFT LEKSFEGNVF MYYGLSNFYQ NHRRYVKSRD DSQLNGDSSA LLNPSKECEP Y RRNEDKPI APCGAIANSM FNDTLELFLI GNDSYPIPIA LKKKGIAWWT DKNVKFRNPP GGDNLEERFK GTTKPVNWLK PV YMLDSDP DNNGFINEDF IVWMRTAALP TFRKLYRLIE RKSDLHPTLP AGRYSLNVTY NYPVHYFDGR KRMILSTISW MGG KNPFLG IAYIAVGSIS FLLGVVLLVI NHKYRNSSNT ADITI

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #6: O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy...

MacromoleculeName: O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy)phosphoryl]-L-serine
type: ligand / ID: 6 / Number of copies: 1 / Formula: 17F
Molecular weightTheoretical: 788.043 Da
Chemical component information

ChemComp-17F:
O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy)phosphoryl]-L-serine / phospholipid*YM

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration7.5 mg/mL
BufferpH: 6.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78800
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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