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- EMDB-23071: Structure of the S. cerevisiae phosphatidylcholine flippase Dnf2-... -

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Basic information

Entry
Database: EMDB / ID: EMD-23071
TitleStructure of the S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
Map dataCryo-EM 3D map of the S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
Sample
  • Complex: S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
    • Protein or peptide: Phospholipid-transporting ATPase DNF2
    • Protein or peptide: Alkylphosphocholine resistance protein LEM3
  • Ligand: CHOLESTEROL
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
  • Ligand: alpha-D-mannopyranose
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsP4 ATPase / Phosphatidylcholine Flippases / TRANSLOCASE
Function / homology
Function and homology information


regulation of vacuole organization / glycosylceramide flippase activity / mating projection tip membrane / aminophospholipid translocation / Ion transport by P-type ATPases / aminophospholipid flippase activity / phosphatidylserine flippase activity / ceramide translocation / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex ...regulation of vacuole organization / glycosylceramide flippase activity / mating projection tip membrane / aminophospholipid translocation / Ion transport by P-type ATPases / aminophospholipid flippase activity / phosphatidylserine flippase activity / ceramide translocation / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / phosphatidylethanolamine flippase activity / phosphatidylcholine floppase activity / cellular bud neck / P-type phospholipid transporter / phospholipid translocation / establishment or maintenance of cell polarity / cell periphery / endocytosis / protein transport / cell surface receptor signaling pathway / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / plasma membrane / cytoplasm
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
Phospholipid-transporting ATPase accessory subunit LEM3 / Phospholipid-transporting ATPase DNF2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsBai L / You Q / Jain BK / Duan HD / Kovach A / Graham TR / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 to H.L. and GM107978 to T.R.G. United States
CitationJournal: Elife / Year: 2020
Title: Transport mechanism of P4 ATPase phosphatidylcholine flippases.
Authors: Lin Bai / Qinglong You / Bhawik K Jain / H Diessel Duan / Amanda Kovach / Todd R Graham / Huilin Li /
Abstract: The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 ...The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 and human ATP8A1-have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the Dnf1-Lem3 and Dnf2-Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.
History
DepositionDec 7, 2020-
Header (metadata) releaseJan 6, 2021-
Map releaseJan 6, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ky8
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23071.png

Downloads & links

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Map

FileDownload / File: emd_23071.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM 3D map of the S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 280 pix.
= 231.28 Å		0.83 Å/pix.
x 280 pix.
= 231.28 Å		0.83 Å/pix.
x 280 pix.
= 231.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.09213252 - 0.14402431
Average (Standard dev.)0.00023420129 (±0.0033276528)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 231.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8260.8260.826
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z231.280231.280231.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0920.1440.000

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Supplemental data

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Sample components

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Entire : S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in t...

EntireName: S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
Components
  • Complex: S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
    • Protein or peptide: Phospholipid-transporting ATPase DNF2
    • Protein or peptide: Alkylphosphocholine resistance protein LEM3
  • Ligand: CHOLESTEROL
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
  • Ligand: alpha-D-mannopyranose
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in t...

SupramoleculeName: S. cerevisiae phosphatidylcholine flippase Dnf2-Lem3 complex in the E1-ATP state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c

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Macromolecule #1: Phospholipid-transporting ATPase DNF2

MacromoleculeName: Phospholipid-transporting ATPase DNF2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 182.834469 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSSPSKPTSP FVDDIEHESG SASNGLSSMS PFDDSFQFEK PSSAHGNIEV AKTGGSVLKR QSKPMKDIST PDLSKVTFDG IDDYSNDND INDDDELNGK KTEIHEHENE VDDDLHSFQA TPMPNTGGFE DVELDNNEGS NNDSQADHKL KRVRFGTRRN K SGRIDINR ...String:
MSSPSKPTSP FVDDIEHESG SASNGLSSMS PFDDSFQFEK PSSAHGNIEV AKTGGSVLKR QSKPMKDIST PDLSKVTFDG IDDYSNDND INDDDELNGK KTEIHEHENE VDDDLHSFQA TPMPNTGGFE DVELDNNEGS NNDSQADHKL KRVRFGTRRN K SGRIDINR SKTLKWAKKN FHNAIDEFST KEDSLENSAL QNRSDELRTV YYNLPLPEDM LDEDGLPLAV YPRNKIRTTK YT PLTFFPK NILFQFHNFA NIYFLILLIL GAFQIFGVTN PGFASVPLIV IVIITAIKDG IEDSRRTVLD LEVNNTRTHI LSG VKNENV AVDNVSLWRR FKKANTRALI KIFEYFSENL TAAGREKKLQ KKREELRRKR NSRSFGPRGS LDSIGSYRMS ADFG RPSLD YENLNQTMSQ ANRYNDGENL VDRTLQPNPE CRFAKDYWKN VKVGDIVRVH NNDEIPADMI LLSTSDVDGA CYVET KNLD GETNLKVRQS LKCSKIIKSS RDITRTKFWV ESEGPHANLY SYQGNFKWQD TQNGNIRNEP VNINNLLLRG CTLRNT KWA MGMVIFTGDD TKIMINAGVT PTKKSRISRE LNFSVILNFV LLFILCFTAG IVNGVYYKQK PRSRDYFEFG TIGGSAS TN GFVSFWVAVI LYQSLVPISL YISVEIIKTA QAIFIYTDVL LYNAKLDYPC TPKSWNISDD LGQIEYIFSD KTGTLTQN V MEFKKCTING VSYGRAYTEA LAGLRKRQGV DVESEGRREK EEIAKDRETM IDELRSMSDN TQFCPEDLTF VSKEIVEDL KGSSGDHQQK CCEHFLLALA LCHSVLVEPN KDDPKKLDIK AQSPDESALV STARQLGYSF VGSSKSGLIV EIQGVQKEFQ VLNVLEFNS SRKRMSCIIK IPGSTPKDEP KALLICKGAD SVIYSRLDRT QNDATLLEKT ALHLEEYATE GLRTLCLAQR E LTWSEYER WVKTYDVAAA SVTNREEELD KVTDVIEREL ILLGGTAIED RLQDGVPDSI ALLAEAGIKL WVLTGDKVET AI NIGFSCN VLNNDMELLV VKASGEDVEE FGSDPIQVVN NLVTKYLREK FGMSGSEEEL KEAKREHGLP QGNFAVIIDG DAL KVALNG EEMRRKFLLL CKNCKAVLCC RVSPAQKAAV VKLVKKTLDV MTLAIGDGSN DVAMIQSADV GVGIAGEEGR QAVM CSDYA IGQFRYVTRL VLVHGKWCYK RLAEMIPQFF YKNVIFTLSL FWYGIYNNFD GSYLFEYTYL TFYNLAFTSV PVILL AVLD QDVSDTVSML VPQLYRVGIL RKEWNQTKFL WYMLDGVYQS VICFFFPYLA YHKNMVVTEN GLGLDHRYFV GVFVTA IAV TSCNFYVFME QYRWDWFCGL FICLSLAVFY GWTGIWTSSS SSNEFYKGAA RVFAQPAYWA VLFVGVLFCL LPRFTID CI RKIFYPKDIE IVREMWLRGD FDLYPQGYDP TDPSRPRINE IRPLTDFKEP ISLDTHFDGV SHSQETIVTE EIPMSILN G EQGSRKGYRV STTLERRDQL SPVTTTNNLP RRSMASARGN KLRTSLDRTR EEMLANHQLD TRYSVERARA SLDLPGINH AETLLSQRSR DR

UniProtKB: Phospholipid-transporting ATPase DNF2

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Macromolecule #2: Alkylphosphocholine resistance protein LEM3

MacromoleculeName: Alkylphosphocholine resistance protein LEM3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 47.490395 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MVNFDLGQVG EVFRRKDKGA IVSGDNPEEE EDVDASEFEE DEVKPVRTKN RRPKEDAFTQ QRLAAINPVL TPRTVLPLYL LIAVVFVIV GGCILAQNSK VDEVTIYYQD CMTNATSSWS DIPSEHWQFV FHKYKTYNTA PQWRFVDDES DDFTKQRGTC Q IRFTTPSD ...String:
MVNFDLGQVG EVFRRKDKGA IVSGDNPEEE EDVDASEFEE DEVKPVRTKN RRPKEDAFTQ QRLAAINPVL TPRTVLPLYL LIAVVFVIV GGCILAQNSK VDEVTIYYQD CMTNATSSWS DIPSEHWQFV FHKYKTYNTA PQWRFVDDES DDFTKQRGTC Q IRFTTPSD MKNNVYLNYV LEKFAANHRR YVLSFSEDQI RGEDASYETV HDATGINCKP LSKNADGKIY YPCGLIANSM FN DTFPLQL TNVGDTSNNY SLTNKGINWE SDKKRYKKTK YNYTQIAPPP YWEKMYPDGY NETNIPDIQD WEEFQNWMRP GAF DKITKL IRINKNDTLP AGEYQLDIGL HWPVLEFNGK KGIYLTHGSH LGGRNPFLGI VYLIGGCICA AMALILLTFW LFGG RKIAD ASSLSWNMK

UniProtKB: Phospholipid-transporting ATPase accessory subunit LEM3

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 5 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: ACP
Molecular weightTheoretical: 505.208 Da
Chemical component information

ChemComp-ACP:
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / AMP-PCP, energy-carrying molecule analogue*YM

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Macromolecule #8: alpha-D-mannopyranose

MacromoleculeName: alpha-D-mannopyranose / type: ligand / ID: 8 / Number of copies: 1 / Formula: MAN
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-MAN:
alpha-D-mannopyranose

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 358687
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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