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- PDB-6roh: Cryo-EM structure of the autoinhibited Drs2p-Cdc50p -

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Basic information

Entry
Database: PDB / ID: 6roh
TitleCryo-EM structure of the autoinhibited Drs2p-Cdc50p
Components
  • Cell division control protein 50
  • Probable phospholipid-transporting ATPase DRS2
KeywordsLIPID TRANSPORT / Lipid Flippase / P-type ATPase / PS Transport.
Function / homology
Function and homology information


Cdc50p-Drs2p complex / actin cortical patch localization / aminophospholipid translocation / phosphatidylcholine flippase activity / Ion transport by P-type ATPases / post-Golgi vesicle-mediated transport / phosphatidylserine flippase activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / ATPase-coupled intramembrane lipid transporter activity ...Cdc50p-Drs2p complex / actin cortical patch localization / aminophospholipid translocation / phosphatidylcholine flippase activity / Ion transport by P-type ATPases / post-Golgi vesicle-mediated transport / phosphatidylserine flippase activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / 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 / Neutrophil degranulation / intracellular protein transport / trans-Golgi network / endocytosis / late endosome membrane / endosome membrane / magnesium ion binding / endoplasmic reticulum / Golgi apparatus / 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 / P-type ATPase, cytoplasmic domain N / 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 / P-type ATPase, cytoplasmic domain N / 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
1,2-DICAPROYL-SN-PHOSPHATIDYL-L-SERINE / 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: 2.8 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.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
Revision 2.1Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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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)212,9777
Polymers211,1032
Non-polymers1,8745
Water362
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10660 Å2
ΔGint-20 kcal/mol
Surface area57940 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: 163730.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: D560 described by Aspartate beryllium trifluoride (BFD) engineered C-terminal GGGG-LVPRGS-BAD-tag Residues 1-104 are removed by proteolysis with thrombin Residues 1364-1460 are removed after ...Details: D560 described by Aspartate beryllium trifluoride (BFD) engineered C-terminal GGGG-LVPRGS-BAD-tag Residues 1-104 are removed by proteolysis with thrombin Residues 1364-1460 are removed after cleaving of the purification tag by thrombin
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DRS2, YAL026C, FUN38 / Plasmid: pYedp60 / Production host: Saccharomyces cerevisiae S288C (yeast)
References: UniProt: P39524, P-type phospholipid transporter
#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)
Strain: ATCC 204508 / S288c / Gene: CDC50, YCR094W, YCR94W / Plasmid: pYedp60 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P25656

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Sugars , 3 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.: 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
#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
#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 4 molecules

#5: Chemical ChemComp-PSF / 1,2-DICAPROYL-SN-PHOSPHATIDYL-L-SERINE / PHOSPHATIDYLSERINE


Mass: 455.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H34NO10P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: 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 protein modificationY

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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 / Type: COMPLEX / 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
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 FIELD
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 60 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
7Cootmodel fitting
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2157578
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 752881 / Symmetry type: POINT

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