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- EMDB-13353: Phophorylated Drs2p-Cdc50p in a PS and ATP-bound E2P state -

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Basic information

Entry
Database: EMDB / ID: EMD-13353
TitlePhophorylated Drs2p-Cdc50p in a PS and ATP-bound E2P state
Map dataSharpened map
Sample
  • Complex: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P and transport substrate PS
    • Protein or peptide: Probable phospholipid-transporting ATPase DRS2
    • Protein or peptide: Cell division control protein 50
  • 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: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: O-[(R)-[(2S)-2-(hexadecanoyloxy)-3-(octadecanoyloxy)propoxy](hydroxy)phosphoryl]-D-serine
  • Ligand: water
Function / homology
Function and homology information


Cdc50p-Drs2p complex / actin cortical patch localization / aminophospholipid translocation / Ion transport by P-type ATPases / phosphatidylcholine flippase activity / post-Golgi vesicle-mediated transport / phosphatidylserine flippase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity ...Cdc50p-Drs2p complex / actin cortical patch localization / aminophospholipid translocation / Ion transport by P-type ATPases / phosphatidylcholine flippase activity / post-Golgi vesicle-mediated transport / phosphatidylserine flippase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylserine floppase activity / phosphatidylethanolamine flippase activity / P-type phospholipid transporter / endocytic recycling / retrograde transport, endosome to Golgi / phosphatidylinositol-4-phosphate binding / phospholipid translocation / 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
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) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...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) / 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 accessory subunit CDC50 / Phospholipid-transporting ATPase DRS2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsTimcenko M / Wang Y / Lyons JA / Nissen P / Lindorff-Larsen K
Funding support Denmark, 1 items
OrganizationGrant numberCountry
LundbeckfondenR155-2015-266 Denmark
CitationJournal: To Be Published
Title: Substrate Transport and Specificity in a Phospholipid Flippase
Authors: Wang Y / Lyons JA / Timcenko M / Kummerer F / de Groot BL / Nissen P / Gapsys V / Lindorff-Larsen K
History
DepositionAug 10, 2021-
Header (metadata) releaseAug 24, 2022-
Map releaseAug 24, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13353.png

Downloads & links

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Map

FileDownload / File: emd_13353.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.0312 Å
Density
Contour LevelBy AUTHOR: 0.19
Minimum - Maximum-0.75901985 - 1.2419199
Average (Standard dev.)0.0009507362 (±0.032908168)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 263.9872 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Map

Fileemd_13353_additional_1.map
AnnotationMap
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_13353_half_map_1.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_13353_half_map_2.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

EntireName: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P and transport substrate PS
Components
  • Complex: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P and transport substrate PS
    • Protein or peptide: Probable phospholipid-transporting ATPase DRS2
    • Protein or peptide: Cell division control protein 50
  • 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: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: O-[(R)-[(2S)-2-(hexadecanoyloxy)-3-(octadecanoyloxy)propoxy](hydroxy)phosphoryl]-D-serine
  • Ligand: water

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

SupramoleculeName: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P and transport substrate PS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288c
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: W303-1A dpep4 / Recombinant plasmid: pYeDP60
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Probable phospholipid-transporting ATPase DRS2

MacromoleculeName: Probable phospholipid-transporting ATPase DRS2 / type: protein_or_peptide / ID: 1
Details: Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus.
Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 154.006766 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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 (PHD)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 ISDSRPHVQQ FQNAIRKVRQ VQRMKKQRGF AFSQAEEGG QEKIVRMYDT TQKRGKYGEL QDASANPFND NNGLGSNDFE SAEPFIENPF ADGNQNSNRF SSSRDDISFD I

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Macromolecule #2: Cell division control protein 50

MacromoleculeName: Cell division control protein 50 / type: protein_or_peptide / ID: 2 / Details: Cdc50p with Thrombin cleavable C-terminal His-tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 45.037312 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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 LREIL

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Macromolecule #5: (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: 5 / 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 #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #8: O-[(R)-[(2S)-2-(hexadecanoyloxy)-3-(octadecanoyloxy)propoxy](hydr...

MacromoleculeName: O-[(R)-[(2S)-2-(hexadecanoyloxy)-3-(octadecanoyloxy)propoxy](hydroxy)phosphoryl]-D-serine
type: ligand / ID: 8 / Number of copies: 1 / Formula: Q3G
Molecular weightTheoretical: 764.022 Da
Chemical component information

ChemComp-Q3G:
O-[(R)-[(2S)-2-(hexadecanoyloxy)-3-(octadecanoyloxy)propoxy](hydroxy)phosphoryl]-D-serine / phospholipid*YM

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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
Component:
ConcentrationFormulaName
50.0 mMMOPS-Tris
100.0 mMKCl
5.0 mMMgCl2
1.0 mMDTTDithiothreitol
0.03 mg/mLLMNGlauryl maltose neopentyl glycol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Incubated with 0.1 mg/mL POPS for 1 hour. 3mM ATP was added to the sample just before application to the grid..
DetailsPurified in detergent lauryl maltose neopentyl glycol (LMNG)

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9837 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3029402
CTF correctionSoftware - Name: cryoSPARC (ver. 2) / Software - details: patch CTF
Startup modelType of model: OTHER / Details: ab initio determined in cryoSPARC v2
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Software - details: non-uniform refinement / Details: non-uniform refinement in cryoSPARC v3 / Number images used: 91415
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Software - details: heterogeneous refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3) / Software - details: non-uniform refinement
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2) / Software - details: heterogeneous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6roj

DetailsMolecular dynamics flexible fitting and energy minimization in Gromacs
RefinementSpace: REAL / Overall B value: 69.1 / Target criteria: correlation coefficient
Output model

PDB-7pem:
Cryo-EM structure of phophorylated Drs2p-Cdc50p in a PS and ATP-bound E2P state

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