+
Open data
-
Basic information
Entry | Database: EMDB / ID: EMD-12896 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Drs2p-Cdc50p in the E1-AMPPCP state with PI4P bound | ||||||||||||
![]() | Sharpened map | ||||||||||||
![]() |
| ||||||||||||
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 / 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 | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||||||||
![]() | Timcenko M / Dieudonne T / Montigny C / Boesen T / Lyons JA / Lenoir G / Nissen P | ||||||||||||
Funding support | ![]() ![]()
| ||||||||||||
![]() | ![]() 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 |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
-
Downloads & links
-EMDB archive
Map data | ![]() | 59.3 MB | ![]() | |
---|---|---|---|---|
Header (meta data) | ![]() ![]() | 26.7 KB 26.7 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 8.9 KB | Display | ![]() |
Images | ![]() | 96 KB | ||
Others | ![]() ![]() | 59.3 MB 59.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7oh7MC ![]() 7oh4C ![]() 7oh5C ![]() 7oh6C C: citing same article ( M: atomic model generated by this map |
---|---|
Similar structure data |
-
Links
EMDB pages | ![]() ![]() |
---|---|
Related items in Molecule of the Month |
-
Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Sharpened map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.0312 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Half map: Half-map B
File | emd_12896_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half-map B | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half-map A
File | emd_12896_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half-map A | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-
Sample components
-Entire : Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P
Entire | Name: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P |
---|---|
Components |
|
-Supramolecule #1: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P
Supramolecule | Name: Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4P type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
---|---|
Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Supramolecule #2: Drs2p
Supramolecule | Name: Drs2p / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
---|---|
Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Supramolecule #3: Cdc50p
Supramolecule | Name: Cdc50p / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
---|---|
Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #1: Probable phospholipid-transporting ATPase DRS2,Probable phospholi...
Macromolecule | Name: Probable phospholipid-transporting ATPase DRS2,Probable phospholipid-transporting ATPase DRS2 type: protein_or_peptide / ID: 1 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. Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter |
---|---|
Source (natural) | Organism: ![]() ![]() Strain: ATCC 204508 / S288c |
Molecular weight | Theoretical: 164.175359 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 ISLVPRGSDS RPHVQQFQNA IRKVRQVQRM KKQRG FAFS QAEEGGQEKI VRMYDTTQKR GKYGELQDAS ANPFNDNNGL GSNDFESAEP FIENPFADGN QNSNRFSSSR DDISFD IGG GGLVPRGSGG TAAAPGPAPA PAPASAPAAA APAGAGTPVT APLAGTIWKV LASEGQTVAA GEVLLILEAM KMETEIR AA QAGTVRGIAV KAGDAVAVGD TLM |
-Macromolecule #2: Cell division control protein 50
Macromolecule | Name: Cell division control protein 50 / type: protein_or_peptide / ID: 2 / Details: Cdc50p with Thrombin cleavable C-terminal His-tag / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: ![]() ![]() Strain: ATCC 204508 / S288c |
Molecular weight | Theoretical: 47.371797 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 LREILGGGGL VPRG SGGHH HHHHHHHH |
-Macromolecule #5: (2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-...
Macromolecule | Name: (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 type: ligand / ID: 5 / Number of copies: 1 / Formula: 2Y5 |
---|---|
Molecular weight | Theoretical: 967.108 Da |
Chemical component information | ![]() ChemComp-2Y5: |
-Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
Macromolecule | Name: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 1 / Formula: ACP |
---|---|
Molecular weight | Theoretical: 505.208 Da |
Chemical component information | ![]() ChemComp-ACP: |
-Macromolecule #7: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG |
---|---|
Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #8: water
Macromolecule | Name: water / type: ligand / ID: 8 / Number of copies: 3 / Formula: HOH |
---|---|
Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
![]() | single particle reconstruction |
Aggregation state | particle |
-
Sample preparation
Concentration | 0.6 mg/mL | ||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7 Component:
| ||||||||||||||||||
Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV | ||||||||||||||||||
Details | Purified in detergent lauryl maltose neopentyl glycol (LMNG) |
-
Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2165 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |