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- PDB-7pem: Cryo-EM structure of phophorylated Drs2p-Cdc50p in a PS and ATP-b... -

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Basic information

Entry
Database: PDB / ID: 7pem
TitleCryo-EM structure of phophorylated Drs2p-Cdc50p in a PS and ATP-bound E2P state
Components
  • Cell division control protein 50
  • Probable phospholipid-transporting ATPase DRS2
KeywordsMEMBRANE PROTEIN / Lipid Flippase / P4 ATPase / trans-Golgi Network / Phosphatidylserine transport
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
Chem-2Y5 / ADENOSINE-5'-TRIPHOSPHATE / Chem-Q3G / Phospholipid-transporting ATPase accessory subunit CDC50 / Phospholipid-transporting ATPase DRS2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsTimcenko, M. / Wang, Y. / Lyons, J.A. / Nissen, P. / Lindorff-Larsen, K.
Funding support Denmark, 1items
OrganizationGrant numberCountry
LundbeckfondenR155-2015-266 Denmark
CitationJournal: To Be Published
Title: Substrate Transport and Specificity in a Phospholipid Flippase
Authors: Wang, Y. / Lyons, J.A. / Timcenko, M. / Kummerer, F. / de Groot, B.L. / Nissen, P. / Gapsys, V. / Lindorff-Larsen, K.
History
DepositionAug 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 24, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Probable phospholipid-transporting ATPase DRS2
C: Cell division control protein 50
hetero molecules


Theoretical massNumber of molelcules
Total (without water)202,9049
Polymers199,0442
Non-polymers3,8607
Water362
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12100 Å2
ΔGint-38 kcal/mol
Surface area61710 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AC

#1: Protein Probable phospholipid-transporting ATPase DRS2


Mass: 154006.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DRS2, YAL026C, FUN38 / Plasmid: pYeDP60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): W303-1A dpep4
References: UniProt: P39524, P-type phospholipid transporter
#2: Protein Cell division control protein 50


Mass: 45037.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cdc50p with Thrombin cleavable C-terminal His-tag
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CDC50, YCR094W, YCR94W / Plasmid: pYeDP60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): W303-1A dpep4 / References: UniProt: P25656

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Sugars , 2 types, 3 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Polysaccharide beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4) ...beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][b-D-Manp]{}}}}}LINUCSPDB-CARE

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Non-polymers , 5 types, 6 molecules

#5: Chemical 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 / Phosphatidylinositol-4-phosphate / PI4P


Mass: 967.108 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H84O16P2
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#8: Chemical ChemComp-Q3G / O-[(R)-[(2S)-2-(hexadecanoyloxy)-3-(octadecanoyloxy)propoxy](hydroxy)phosphoryl]-D-serine


Mass: 764.022 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H78NO10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P and transport substrate PS
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288c
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: W303-1A dpep4 / Plasmid: pYeDP60
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMMOPS-Tris1
2100 mMKCl1
35 mMMgCl21
41 mMDithiothreitolDTT1
50.03 mg/mLlauryl maltose neopentyl glycolLMNG1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified in detergent lauryl maltose neopentyl glycol (LMNG)
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Incubated with 0.1 mg/mL POPS for 1 hour. 3mM ATP was added to the sample just before application to the grid.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9837
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc7_3834refinement
PHENIX1.18rc7_3834refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC2particle selection
2EPUimage acquisition
4cryoSPARC2CTF correctionpatch CTF
7PyMOLmodel fitting
8Cootmodel fitting
10cryoSPARC2initial Euler assignmentheterogeneous refinement
11cryoSPARC3final Euler assignmentnon-uniform refinement
12cryoSPARC2classificationheterogeneous refinement
13cryoSPARC33D reconstructionnon-uniform refinement
14PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3029402
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91415 / Details: non-uniform refinement in cryoSPARC v3 / Symmetry type: POINT
Atomic model buildingB value: 69.1 / Space: REAL / Target criteria: correlation coefficient
Details: Molecular dynamics flexible fitting and energy minimization in Gromacs
Atomic model buildingPDB-ID: 6ROJ

6roj

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 61.24 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001811883
ELECTRON MICROSCOPYf_angle_d0.441116107
ELECTRON MICROSCOPYf_chiral_restr0.03991836
ELECTRON MICROSCOPYf_plane_restr0.00291999
ELECTRON MICROSCOPYf_dihedral_angle_d22.30461655

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