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- EMDB-4974: Cryo-EM structure of the activated Drs2p-Cdc50p -

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Basic information

Entry
Database: EMDB / ID: EMD-4974
TitleCryo-EM structure of the activated Drs2p-Cdc50p
Map data
Sample
  • Complex: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P
    • Protein or peptide: Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decarboxylase alpha chain
    • Protein or peptide: Cell division control protein 50
  • Ligand: MAGNESIUM ION
  • Ligand: (2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate
  • Ligand: water
Function / homology
Function and homology information


oxaloacetate decarboxylase (Na+ extruding) / decarboxylation-driven active transmembrane transporter activity / Cdc50p-Drs2p complex / actin cortical patch localization / Ion transport by P-type ATPases / aminophospholipid translocation / phosphatidylcholine flippase activity / post-Golgi vesicle-mediated transport / oxaloacetate decarboxylase activity / phosphatidylserine flippase activity ...oxaloacetate decarboxylase (Na+ extruding) / decarboxylation-driven active transmembrane transporter activity / Cdc50p-Drs2p complex / actin cortical patch localization / Ion transport by P-type ATPases / aminophospholipid translocation / phosphatidylcholine flippase activity / post-Golgi vesicle-mediated transport / oxaloacetate decarboxylase activity / phosphatidylserine flippase activity / phosphatidylserine floppase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylethanolamine flippase activity / endocytic recycling / P-type phospholipid transporter / phosphatidylinositol-4-phosphate binding / retrograde transport, endosome to Golgi / phospholipid translocation / sodium ion transport / Neutrophil degranulation / intracellular protein transport / trans-Golgi network / endocytosis / late endosome membrane / endosome membrane / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / plasma membrane / cytosol
Similarity search - Function
Oxaloacetate decarboxylase, alpha subunit / Carboxylase, conserved domain / Conserved carboxylase domain / Pyruvate carboxyltransferase / HMGL-like / Pyruvate carboxyltransferase domain. / CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal ...Oxaloacetate decarboxylase, alpha subunit / Carboxylase, conserved domain / Conserved carboxylase domain / Pyruvate carboxyltransferase / HMGL-like / Pyruvate carboxyltransferase 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 / Biotin-binding site / Biotin-requiring enzymes attachment site. / Biotin-requiring enzyme / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / Single hybrid motif / Cation transport ATPase (P-type) / 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 / Aldolase-type TIM barrel
Similarity search - Domain/homology
Oxaloacetate decarboxylase alpha chain / Phospholipid-transporting ATPase accessory subunit CDC50 / Phospholipid-transporting ATPase DRS2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast) / Klebsiella pneumoniae (bacteria) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsTimcenko M / Lyons JA / Januliene D / Ulstrup JJ / Dieudonne T / Montigny C / Ash MR / Karlsen JL / Boesen T / Kuhlbrandt W ...Timcenko M / Lyons JA / Januliene D / Ulstrup JJ / Dieudonne T / Montigny C / Ash MR / Karlsen JL / Boesen T / Kuhlbrandt W / Lenoir G / Moeller A / Nissen P
Funding support Denmark, France, 6 items
OrganizationGrant numberCountry
Danish Council for Independent Research0602-02912B Denmark
LundbeckfondenR209-2015-2704 Denmark
LundbeckfondenR171-2014-663 Denmark
French National Research AgencyANR-14-CE09-0022 France
Lundbeckfonden2015-2666 Denmark
French Infrastructure for Integrated Structural BiologyANR-10-INSB-05 France
CitationJournal: Nature / Year: 2019
Title: Structure and autoregulation of a P4-ATPase lipid flippase.
Authors: Milena Timcenko / Joseph A Lyons / Dovile Januliene / Jakob J Ulstrup / Thibaud Dieudonné / Cédric Montigny / Miriam-Rose Ash / Jesper Lykkegaard Karlsen / Thomas Boesen / Werner ...Authors: Milena Timcenko / Joseph A Lyons / Dovile Januliene / Jakob J Ulstrup / Thibaud Dieudonné / Cédric Montigny / Miriam-Rose Ash / Jesper Lykkegaard Karlsen / Thomas Boesen / Werner Kühlbrandt / Guillaume Lenoir / Arne Moeller / Poul Nissen /
Abstract: Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The ...Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.
History
DepositionMay 13, 2019-
Header (metadata) releaseJun 19, 2019-
Map releaseJul 3, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6roj
  • Surface level: 0.013
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4974.png

Downloads & links

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Map

FileDownload / File: emd_4974.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.077 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.05881711 - 0.117117204
Average (Standard dev.)0.00016436083 (±0.0024119904)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 275.712 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0771.0771.077
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z275.712275.712275.712
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0590.1170.000

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Supplemental data

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Mask #1

Fileemd_4974_msk_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Additional map: #1

Fileemd_4974_additional.map
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Half map: #2

Fileemd_4974_half_map_1.map
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Half map: #1

Fileemd_4974_half_map_2.map
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Sample components

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Entire : Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P

EntireName: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P
Components
  • Complex: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P
    • Protein or peptide: Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decarboxylase alpha chain
    • Protein or peptide: Cell division control protein 50
  • Ligand: MAGNESIUM ION
  • Ligand: (2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate
  • Ligand: water

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Supramolecule #1: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P

SupramoleculeName: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: C-terminus removed by proteolytic cleavage of an engineered site.
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast) / Recombinant plasmid: pYedp60

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Macromolecule #1: Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decar...

MacromoleculeName: Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decarboxylase alpha chain
type: protein_or_peptide / ID: 1
Details: Residues 1-104 removed after proteolysis with thrombin Residues 1252-1364 removed after proteolysis with thrombin 1248-1253 - added an addition thrombin cleavage site D560 described by ...Details: Residues 1-104 removed after proteolysis with thrombin Residues 1252-1364 removed after proteolysis with thrombin 1248-1253 - added an addition thrombin cleavage site D560 described by Aspartate beryllium trifluoride (BFD) engineered C-terminal GGGG-LVPRGS-BAD-ta,Residues 1-104 removed after proteolysis with thrombin Residues 1252-1364 removed after proteolysis with thrombin 1248-1253 - added an addition thrombin cleavage site D560 described by Aspartate beryllium trifluoride (BFD) engineered C-terminal GGGG-LVPRGS-BAD-ta
Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 164.240359 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MNDDRETPPK RKPGEDDTLF DIDFLDDTTS HSGSRSKVTN SHANANYIPP SHVLPEETID LDADDDNIEN DVHENLFMSN NHDDQTSWN ANRFDSDAYQ PQSLRAVKPP GLFARFGNGL KNAFTFKRKK GPESFEMNHY NAVTNNELDD NYLDSRNKFN I KILFNRYI ...String:
MNDDRETPPK RKPGEDDTLF DIDFLDDTTS HSGSRSKVTN SHANANYIPP SHVLPEETID LDADDDNIEN DVHENLFMSN NHDDQTSWN ANRFDSDAYQ PQSLRAVKPP GLFARFGNGL KNAFTFKRKK GPESFEMNHY NAVTNNELDD NYLDSRNKFN I KILFNRYI LRKNVGDAEG NGEPRVIHIN DSLANSSFGY SDNHISTTKY NFATFLPKFL FQEFSKYANL FFLCTSAIQQ VP HVSPTNR YTTIGTLLVV LIVSAMKECI EDIKRANSDK ELNNSTAEIF SEAHDDFVEK RWIDIRVGDI IRVKSEEPIP ADT IILSSS EPEGLCYIET ANLDGETNLK IKQSRVETAK FIDVKTLKNM NGKVVSEQPN SSLYTYEGTM TLNDRQIPLS PDQM ILRGA TLRNTAWIFG LVIFTGHETK LLRNATATPI KRTAVEKIIN RQIIALFTVL IVLILISSIG NVIMSTADAK HLSYL YLEG TNKAGLFFKD FLTFWILFSN LVPISLFVTV ELIKYYQAFM IGSDLDLYYE KTDTPTVVRT SSLVEELGQI EYIFS (BFD)KTG TLTRNIMEFK SCSIAGHCYI DKIPEDKTAT VEDGIEVGYR KFDDLKKKLN DPSDEDSPII NDFLTLLATC HT VIPEFQS DGSIKYQAAS PDEGALVQGG ADLGYKFIIR KPNSVTVLLE ETGEEKEYQL LNICEFNSTR KRMSAIFRFP DGS IKLFCK GADTVILERL DDEANQYVEA TMRHLEDYAS EGLRTLCLAM RDISEGEYEE WNSIYNEAAT TLDNRAEKLD EAAN LIEKN LILIGATAIE DKLQDGVPET IHTLQEAGIK IWVLTGDRQE TAINIGMSCR LLSEDMNLLI INEETRDDTE RNLLE KINA LNEHQLSTHD MNTLALVIDG KSLGFALEPE LEDYLLTVAK LCKAVICCRV SPLQKALVVK MVKRKSSSLL LAIGDG AND VSMIQAAHVG VGISGMEGMQ AARSADIAVG QFKFLKKLLL VHGSWSYQRI SVAILYSFYK NTALYMTQFW YVFANAF SG QSIMESWTMS FYNLFFTVWP PFVIGVFDQF VSSRLLERYP QLYKLGQKGQ FFSVYIFWGW IINGFFHSAI VFIGTILI Y RYGFALNMHG ELADHWSWGV TVYTTSVIIV LGKAALVTNQ WTKFTLIAIP GSLLFWLIFF PIYASIFPHA NISREYYGV VKHTYGSGVF WLTLIVLPIF ALVRDFLWKY YKRMYEPETY HVIQEMQKYN ISLVPRGSDS RPHVQQFQNA IRKVRQVQRM KKQRGFAFS QAEEGGQEKI VRMYDTTQKR GKYGELQDAS ANPFNDNNGL GSNDFESAEP FIENPFADGN QNSNRFSSSR D DISFDIGG GGLVPRGSGG TAAAPGPAPA PAPASAPAAA APAGAGTPVT APLAGTIWKV LASEGQTVAA GEVLLILEAM KM ETEIRAA QAGTVRGIAV KAGDAVAVGD TLM

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Macromolecule #2: Cell division control protein 50

MacromoleculeName: Cell division control protein 50 / type: protein_or_peptide / ID: 2 / Details: engineered C-terminal GGGG-LVPRGS-GG-10xHistag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 47.371797 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MVSLFKRGKA PPLTKEGPTS KKPPNTAFRQ QRLKAWQPIL SPQSVLPLLI FVACIFTPIG IGLIVSATKV QDLTIDYSHC DTKASTTAF EDIPKKYIKY HFKSKVENKP QWRLTENENG EQSCELQFEI PNDIKKSIFI YYKITNFYQN HRRYVQSFDT K QILGEPIK ...String:
MVSLFKRGKA PPLTKEGPTS KKPPNTAFRQ QRLKAWQPIL SPQSVLPLLI FVACIFTPIG IGLIVSATKV QDLTIDYSHC DTKASTTAF EDIPKKYIKY HFKSKVENKP QWRLTENENG EQSCELQFEI PNDIKKSIFI YYKITNFYQN HRRYVQSFDT K QILGEPIK KDDLDTSCSP IRSREDKIIY PCGLIANSMF NDTFSQVLSG IDDTEDYNLT NKHISWSIDR HRFKTTKYNA SD IVPPPNW MKKYPDGYTD ENLPDIHTWE EFQVWMRTAA FPKFYKLTLK NESASLPKGK YQMNIELNYP ISLFGGTKSF VLT TNGAIG GRNMSLGVLY LIVAGLCALF GIIFLVKLIF QPRAMGDHTY LNFDDEENED YEDVHAENTT LREILGGGGL VPRG SGGHH HHHHHHHH

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: (2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-...

MacromoleculeName: (2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate
type: ligand / ID: 6 / Number of copies: 1 / Formula: 2Y5
Molecular weightTheoretical: 967.108 Da
Chemical component information

ChemComp-2Y5:
(2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration0.6 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: 278 K / Instrument: FEI VITROBOT MARK IV
Details1mM beryllium fluoride 0.1 mg/mL Brain PI4P in DDM

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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
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average exposure time: 8.0 sec. / Average electron dose: 56.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1047615
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER / Details: ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 418512
FSC plot (resolution estimation)

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