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- EMDB-20467: Cryo-EM structure of S. cerevisiae Drs2p-Cdc50p in the PI4P-activ... -

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Basic information

Entry
Database: EMDB / ID: EMD-20467
TitleCryo-EM structure of S. cerevisiae Drs2p-Cdc50p in the PI4P-activated form
Map dataS. cerevisiae Drs2p-Cdc50p in the PI4P-activated form
Sample
  • Complex: Drs2p-Cdc50p complex
    • Protein or peptide: Probable phospholipid-transporting ATPase DRS2
    • Protein or peptide: Cell division control protein 50
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordscomplex / phospholipid flippase / P-type ATPase / TRANSLOCASE
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 / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / 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 / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / 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 W303 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBai L / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01-CA231466 United States
CitationJournal: Nat Commun / Year: 2019
Title: Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p.
Authors: Lin Bai / Amanda Kovach / Qinglong You / Hao-Chi Hsu / Gongpu Zhao / Huilin Li /
Abstract: The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is ...The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.
History
DepositionJul 14, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 25, 2019-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6psx
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20467.png

Downloads & links

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Map

FileDownload / File: emd_20467.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationS. cerevisiae Drs2p-Cdc50p in the PI4P-activated form
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 240 pix.
= 246.96 Å		1.03 Å/pix.
x 240 pix.
= 246.96 Å		1.03 Å/pix.
x 240 pix.
= 246.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.029 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.099393606 - 0.1590821
Average (Standard dev.)0.00035867695 (±0.0034563164)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 246.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0291.0291.029
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z246.960246.960246.960
α/β/γ90.00090.00090.000
start NX/NY/NZ1548552
NX/NY/NZ198170217
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0990.1590.000

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Supplemental data

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Sample components

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Entire : Drs2p-Cdc50p complex

EntireName: Drs2p-Cdc50p complex
Components
  • Complex: Drs2p-Cdc50p complex
    • Protein or peptide: Probable phospholipid-transporting ATPase DRS2
    • Protein or peptide: Cell division control protein 50
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Drs2p-Cdc50p complex

SupramoleculeName: Drs2p-Cdc50p complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)

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Macromolecule #1: Probable phospholipid-transporting ATPase DRS2

MacromoleculeName: Probable phospholipid-transporting ATPase DRS2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast) / Strain: W303
Molecular weightTheoretical: 153.926781 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (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 EYIFSD KTG TLTRNIMEFK SCSIAGHCYI DKIPEDKTAT VEDGIEVGYR KFDDLKKKLN DPSDEDSPII NDFLTLLATC HTVIPEF QS DGSIKYQAAS PDEGALVQGG ADLGYKFIIR KPNSVTVLLE ETGEEKEYQL LNICEFNSTR KRMSAIFRFP DGSIKLFC K GADTVILERL DDEANQYVEA TMRHLEDYAS EGLRTLCLAM RDISEGEYEE WNSIYNEAAT TLDNRAEKLD EAANLIEKN LILIGATAIE DKLQDGVPET IHTLQEAGIK IWVLTGDRQE TAINIGMSCR LLSEDMNLLI INEETRDDTE RNLLEKINAL NEHQLSTHD MNTLALVIDG KSLGFALEPE LEDYLLTVAK LCKAVICCRV SPLQKALVVK MVKRKSSSLL LAIGDGANDV S MIQAAHVG VGISGMEGMQ AARSADIAVG QFKFLKKLLL VHGSWSYQRI SVAILYSFYK NTALYMTQFW YVFANAFSGQ SI MESWTMS FYNLFFTVWP PFVIGVFDQF VSSRLLERYP QLYKLGQKGQ FFSVYIFWGW IINGFFHSAI VFIGTILIYR YGF ALNMHG ELADHWSWGV TVYTTSVIIV LGKAALVTNQ WTKFTLIAIP GSLLFWLIFF PIYASIFPHA NISREYYGVV KHTY GSGVF WLTLIVLPIF ALVRDFLWKY YKRMYEPETY HVIQEMQKYN ISDSRPHVQQ FQNAIRKVRQ VQRMKKQRGF AFSQA EEGG QEKIVRMYDT TQKRGKYGEL QDASANPFND NNGLGSNDFE SAEPFIENPF ADGNQNSNRF SSSRDDISFD I

UniProtKB: Phospholipid-transporting ATPase DRS2

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Macromolecule #2: Cell division control protein 50

MacromoleculeName: Cell division control protein 50 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast) / Strain: W303
Molecular weightTheoretical: 45.037312 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (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

UniProtKB: Phospholipid-transporting ATPase accessory subunit CDC50

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / 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

BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.0 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

Particle selectionNumber selected: 1126540
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: Coot, UCSF Chimera) / Number images used: 498745
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6psx:
Cryo-EM structure of S. cerevisiae Drs2p-Cdc50p in the PI4P-activated form

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