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- PDB-7oh4: Cryo-EM structure of Drs2p-Cdc50p in the E1 state with PI4P and M... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7oh4 | ||||||||||||
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Title | Cryo-EM structure of Drs2p-Cdc50p in the E1 state with PI4P and Mg2+ bound | ||||||||||||
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![]() | MEMBRANE PROTEIN / Lipid Flippase / P4 ATPase / trans-Golgi Network / Phosphatidylserine transport | ||||||||||||
Function / homology | ![]() 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 / endocytic recycling / P-type phospholipid transporter / 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 | ||||||||||||
Biological species | ![]() ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | ||||||||||||
![]() | Timcenko, M. / Dieudonne, T. / Montigny, C. / Boesen, T. / Lyons, J.A. / Lenoir, G. / Nissen, P. | ||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() Title: Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase. Authors: Milena Timcenko / Thibaud Dieudonné / Cédric Montigny / Thomas Boesen / Joseph A Lyons / Guillaume Lenoir / Poul Nissen / ![]() ![]() Abstract: P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of ...P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na/K-ATPase or Ca-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent. | ||||||||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 280.3 KB | Display | ![]() |
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PDB format | ![]() | 218.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 12893MC ![]() 7oh5C ![]() 7oh6C ![]() 7oh7C C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 2 types, 2 molecules AC
#1: Protein | Mass: 164175.359 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.,Drs2p with a Thrombin cleavable C-terminal biotin ...Details: Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus.,Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus. Source: (gene. exp.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: DRS2, YAL026C, FUN38 / Plasmid: pYeDP60 / Production host: ![]() ![]() References: UniProt: P39524, P-type phospholipid transporter |
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#2: Protein | Mass: 47371.797 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.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: CDC50, YCR094W, YCR94W / Plasmid: pYeDP60 / Production host: ![]() ![]() |
-Sugars , 3 types, 4 molecules ![](data/chem/img/NAG.gif)
#3: Polysaccharide | Source method: isolated from a genetically manipulated source #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 | Source method: isolated from a genetically manipulated source #7: Sugar | ChemComp-NAG / | |
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-Non-polymers , 3 types, 5 molecules ![](data/chem/img/2Y5.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/HOH.gif)
![](data/chem/img/MG.gif)
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#5: Chemical | ChemComp-2Y5 / ( |
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#6: Chemical | ChemComp-MG / |
#8: Water | ChemComp-HOH / |
-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Source (recombinant) |
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Buffer solution | pH: 7 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm |
Image recording | Average 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: 2074 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
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Processing
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 780933 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 167226 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 55 / Space: REAL / Target criteria: correlation coefficient Details: Domains were rigid body fit into the density and manually adjusted and reconnected. | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6ROJ | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 116.47 Å2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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