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- EMDB-13711: Cryo-EM structure of ATP8B1-CDC50A in E2P autoinhibited state -

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Basic information

Entry
Database: EMDB / ID: EMD-13711
TitleCryo-EM structure of ATP8B1-CDC50A in E2P autoinhibited state
Map dataMap of ATP8B1-CDC50A E2P autoinhibited at 3.1A, sharpened at -84A^2 B factor. Data obtained in the presence of 1mM BeF.
Sample
  • Complex: ATP8B1-CDC50A
    • Protein or peptide: Phospholipid-transporting ATPase IC
    • Protein or peptide: Cell cycle control protein 50A
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


vestibulocochlear nerve formation / regulation of plasma membrane organization / regulation of microvillus assembly / positive regulation of phospholipid translocation / phosphatidylcholine flippase activity / aminophospholipid flippase activity / aminophospholipid transport / phosphatidylserine flippase activity / protein localization to endosome / inner ear receptor cell development ...vestibulocochlear nerve formation / regulation of plasma membrane organization / regulation of microvillus assembly / positive regulation of phospholipid translocation / phosphatidylcholine flippase activity / aminophospholipid flippase activity / aminophospholipid transport / phosphatidylserine flippase activity / protein localization to endosome / inner ear receptor cell development / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity / positive regulation of protein exit from endoplasmic reticulum / xenobiotic transmembrane transport / cardiolipin binding / apical protein localization / phosphatidylcholine floppase activity / stereocilium / bile acid metabolic process / bile acid and bile salt transport / P-type phospholipid transporter / phospholipid translocation / transport vesicle membrane / azurophil granule membrane / Golgi organization / Ion transport by P-type ATPases / specific granule membrane / monoatomic ion transmembrane transport / regulation of chloride transport / sensory perception of sound / trans-Golgi network / positive regulation of neuron projection development / late endosome membrane / early endosome membrane / nuclear body / apical plasma membrane / negative regulation of DNA-templated transcription / Neutrophil degranulation / structural molecule activity / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / nucleoplasm / ATP binding / membrane / plasma membrane / cytosol
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 IC / Cell cycle control protein 50A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDieudonne T / Abad-Herrera S / Juknaviciute Laursen M / Lejeune M / Stock C / Slimani K / Jaxel C / Lyons JA / Montigny C / Gunther Pomorski T ...Dieudonne T / Abad-Herrera S / Juknaviciute Laursen M / Lejeune M / Stock C / Slimani K / Jaxel C / Lyons JA / Montigny C / Gunther Pomorski T / Nissen P / Lenoir G
Funding support France, 6 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-14-CE09-0022 France
H2020 Marie Curie Actions of the European Commission101024542 France
LundbeckfondenR310-2018-3713 France
German Research Foundation (DFG)GU 1133/11-1 France
European Molecular Biology Organization (EMBO)7881 France
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INSB-05 France
CitationJournal: Elife / Year: 2022
Title: Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders.
Authors: Thibaud Dieudonné / Sara Abad Herrera / Michelle Juknaviciute Laursen / Maylis Lejeune / Charlott Stock / Kahina Slimani / Christine Jaxel / Joseph A Lyons / Cédric Montigny / Thomas ...Authors: Thibaud Dieudonné / Sara Abad Herrera / Michelle Juknaviciute Laursen / Maylis Lejeune / Charlott Stock / Kahina Slimani / Christine Jaxel / Joseph A Lyons / Cédric Montigny / Thomas Günther Pomorski / Poul Nissen / Guillaume Lenoir /
Abstract: P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with ...P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.
History
DepositionOct 8, 2021-
Header (metadata) releaseApr 27, 2022-
Map releaseApr 27, 2022-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13711.png

Downloads & links

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Map

FileDownload / File: emd_13711.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of ATP8B1-CDC50A E2P autoinhibited at 3.1A, sharpened at -84A^2 B factor. Data obtained in the presence of 1mM BeF.
Voxel sizeX=Y=Z: 0.66 Å
Density
Contour LevelBy AUTHOR: 5.5
Minimum - Maximum-29.09944 - 44.12814
Average (Standard dev.)-3.245114e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 274.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13711_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of ATP8B1-CDC50A E2P autoinhibited used...

Fileemd_13711_half_map_1.map
AnnotationHalf map A of ATP8B1-CDC50A E2P autoinhibited used during refinement and FSC gold-standard calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of ATP8B1-CDC50A E2P autoinhibited used...

Fileemd_13711_half_map_2.map
AnnotationHalf map B of ATP8B1-CDC50A E2P autoinhibited used during refinement and FSC gold-standard calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ATP8B1-CDC50A

EntireName: ATP8B1-CDC50A
Components
  • Complex: ATP8B1-CDC50A
    • Protein or peptide: Phospholipid-transporting ATPase IC
    • Protein or peptide: Cell cycle control protein 50A
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: ATP8B1-CDC50A

SupramoleculeName: ATP8B1-CDC50A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: W303 1b
Molecular weightTheoretical: 186 KDa

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Macromolecule #1: Phospholipid-transporting ATPase IC

MacromoleculeName: Phospholipid-transporting ATPase IC / 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: 144.182062 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GGGGGMSTER DSETTFDEDS QPNDEVVPYS DDETEDELDD QGSAVEPEQN RVNREAEENR EPFRKECTWQ VKANDRKYHE QPHFMNTKF LCIKESKYAN NAIKTYKYNA FTFIPMNLFE QFKRAANLYF LALLILQAVP QISTLAWYTT LVPLLVVLGV T AIKDLVDD ...String:
GGGGGMSTER DSETTFDEDS QPNDEVVPYS DDETEDELDD QGSAVEPEQN RVNREAEENR EPFRKECTWQ VKANDRKYHE QPHFMNTKF LCIKESKYAN NAIKTYKYNA FTFIPMNLFE QFKRAANLYF LALLILQAVP QISTLAWYTT LVPLLVVLGV T AIKDLVDD VARHKMDKEI NNRTCEVIKD GRFKVAKWKE IQVGDVIRLK KNDFVPADIL LLSSSEPNSL CYVETAELDG ET NLKFKMS LEITDQYLQR EDTLATFDGF IECEEPNNRL DKFTGTLFWR NTSFPLDADK ILLRGCVIRN TDFCHGLVIF AGA DTKIMK NSGKTRFKRT KIDYLMNYMV YTIFVVLILL SAGLAIGHAY WEAQVGNSSW YLYDGEDDTP SYRGFLIFWG YIIV LNTMV PISLYVSVEV IRLGQSHFIN WDLQMYYAEK DTPAKARTTT LNEQLGQIHY IFSDKTGTLT QNIMTFKKCC INGQI YGDH RDASQHNHNK IEQVDFSWNT YADGKLAFYD HYLIEQIQSG KEPEVRQFFF LLAVCHTVMV DRTDGQLNYQ AASPDE GAL VNAARNFGFA FLARTQNTIT ISELGTERTY NVLAILDFNS DRKRMSIIVR TPEGNIKLYC KGADTVIYER LHRMNPT KQ ETQDALDIFA NETLRTLCLC YKEIEEKEFT EWNKKFMAAS VASTNRDEAL DKVYEEIEKD LILLGATAIE DKLQDGVP E TISKLAKADI KIWVLTGDKK ETAENIGFAC ELLTEDTTIC YGEDINSLLH ARMENQRNRG GVYAKFAPPV QESFFPPGG NRALIITGSW LNEILLEKKT KRNKILKLKF PRTEEERRMR TQSKRRLEAK KEQRQKNFVD LACECSAVIC CRVTPKQKAM VVDLVKRYK KAITLAIGDG ANDVNMIKTA HIGVGISGQE GMQAVMSSDY SFAQFRYLQR LLLVHGRWSY IRMCKFLRYF F YKNFAFTL VHFWYSFFNG YSAQTAYEDW FITLYNVLYT SLPVLLMGLL DQDVSDKLSL RFPGLYIVGQ RDLLFNYKRF FV SLLHGVL TSMILFFIPL GAYLQTVGQD GEAPSDYQSF AVTIASALVI TVNFQIGLDT SYWTFVNAFS IFGSIALYFG IMF DFHSAG IHVLFPSAFQ FTGTASNALR QPYIWLTIIL TVAVCLLPVV AIRFLSMTIW PSESDKIQKH RKRLKAEEQW QRRQ QVFRR GVSTRRSAYA FSHQRGYADL ISSGRSIRKK RSPLDAIVAD GTAEYRRTGD S

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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: 37.331832 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: TRRPDNTAFK QQRLPAWQPI LTAGTVLPIF FIIGLIFIPI GIGIFVTSNN IREIEIDYTG TEPSSPCNKC LSPDVTPCFC TINFTLEKS FEGNVFMYYG LSNFYQNHRR YVKSRDDSQL NGDSSALLNP SKECEPYRRN EDKPIAPCGA IANSMFNDTL E LFLIGNDS ...String:
TRRPDNTAFK QQRLPAWQPI LTAGTVLPIF FIIGLIFIPI GIGIFVTSNN IREIEIDYTG TEPSSPCNKC LSPDVTPCFC TINFTLEKS FEGNVFMYYG LSNFYQNHRR YVKSRDDSQL NGDSSALLNP SKECEPYRRN EDKPIAPCGA IANSMFNDTL E LFLIGNDS YPIPIALKKK GIAWWTDKNV KFRNPPGGDN LEERFKGTTK PVNWLKPVYM LDSDPDNNGF INEDFIVWMR TA ALPTFRK LYRLIERKSD LHPTLPAGRY SLNVTYNYPV HYFDGRKRMI LSTISWMGGK NPFLGIAYIA VGSISFLLGV VLL VINHKY R

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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: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #6: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 6 / Number of copies: 4 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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 / N-Acetylglucosamine

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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

Concentration1 mg/mL
BufferpH: 7
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 15mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 605325
CTF correctionSoftware - Name: cryoSPARC (ver. 3)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Details: FSC calculated with mask / Number images used: 104643
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6roh

chain_id: A

6k7l

chain_id: B
RefinementSpace: REAL
Output model

PDB-7py4:
Cryo-EM structure of ATP8B1-CDC50A in E2P autoinhibited state

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