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- PDB-6roj: Cryo-EM structure of the activated Drs2p-Cdc50p -

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Basic information

Entry
Database: PDB / ID: 6roj
TitleCryo-EM structure of the activated Drs2p-Cdc50p
Components
  • Cell division control protein 50
  • Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decarboxylase alpha chain
KeywordsLIPID TRANSPORT / Lipid Flippase / P-type ATPase / PS Transport.
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
Chem-2Y5 / Oxaloacetate decarboxylase alpha chain / Phospholipid-transporting ATPase accessory subunit CDC50 / Phospholipid-transporting ATPase DRS2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsTimcenko, M. / Lyons, J.A. / Januliene, D. / Ulstrup, J.J. / Dieudonne, T. / Montigny, C. / Ash, M.R. / Karlsen, J.L. / Boesen, T. / Kuhlbrandt, W. ...Timcenko, M. / Lyons, J.A. / Januliene, D. / Ulstrup, J.J. / Dieudonne, T. / Montigny, C. / Ash, M.R. / Karlsen, J.L. / Boesen, T. / Kuhlbrandt, W. / Lenoir, G. / Moeller, A. / Nissen, P.
Funding support Denmark, France, 6items
OrganizationGrant numberCountry
Danish Council for Independent Research0602-02912B Denmark
LundbeckfondenR171-2014-663 Denmark
LundbeckfondenR209-2015-2704 Denmark
Lundbeckfonden2015-2666 Denmark
French National Research AgencyANR-14-CE09-0022 France
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jul 31, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decarboxylase alpha chain
C: Cell division control protein 50
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,5257
Polymers211,6122
Non-polymers2,9135
Water362
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10890 Å2
ΔGint-30 kcal/mol
Surface area58830 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AC

#1: Protein Probable phospholipid-transporting ATPase DRS2,Oxaloacetate decarboxylase alpha chain


Mass: 164240.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast), (gene. exp.) Klebsiella pneumoniae (bacteria)
Gene: DRS2, YAL026C, FUN38, oadA / Plasmid: pYedp60 / Production host: Saccharomyces cerevisiae S288C (yeast)
References: UniProt: P39524, UniProt: P13187, P-type phospholipid transporter, oxaloacetate decarboxylase (Na+ extruding)
#2: Protein Cell division control protein 50


Mass: 47371.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: engineered C-terminal GGGG-LVPRGS-GG-10xHistag
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: CDC50, YCR094W, YCR94W / Plasmid: pYedp60 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P25656

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Sugars , 2 types, 3 molecules

#3: 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.: 2
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
#4: 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.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE

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Non-polymers , 3 types, 4 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: 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 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water / Water


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

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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
Type: COMPLEX
Details: C-terminus removed by proteolytic cleavage of an engineered site.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae S288C (yeast) / Plasmid: pYedp60
Buffer solutionpH: 7
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 1mM beryllium fluoride 0.1 mg/mL Brain PI4P in DDM
Specimen supportDetails: 15mA / Grid 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: 278 K

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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 FIELDBright-field microscopy
Image recordingAverage exposure time: 8 sec. / Electron dose: 56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1047615
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 418512 / Symmetry type: POINT

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