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- EMDB-30829: Cryo-EM structure of Dnf1 from Saccharomyces cerevisiae in 90PS w... -

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Basic information

Entry
Database: EMDB / ID: EMD-30829
TitleCryo-EM structure of Dnf1 from Saccharomyces cerevisiae in 90PS with beryllium fluoride (E2P state)
Map data
Sample
  • Complex: Dnf1_Lem3 complex
    • Protein or peptide: Phospholipid-transporting ATPase DNF1
    • Protein or peptide: Alkylphosphocholine resistance protein LEM3
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
Function / homology
Function and homology information


regulation of vacuole organization / glycosylceramide flippase activity / mating projection tip membrane / aminophospholipid translocation / phosphatidylcholine flippase activity / phosphatidylserine flippase activity / phosphatidylserine floppase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylcholine floppase activity ...regulation of vacuole organization / glycosylceramide flippase activity / mating projection tip membrane / aminophospholipid translocation / phosphatidylcholine flippase activity / phosphatidylserine flippase activity / phosphatidylserine floppase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / phosphatidylcholine floppase activity / phosphatidylethanolamine flippase activity / cell septum / cellular bud neck / P-type phospholipid transporter / phospholipid translocation / establishment or maintenance of cell polarity / cell periphery / intracellular protein transport / endocytosis / cell surface receptor signaling pathway / endosome membrane / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein 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 DNF1 / Phospholipid-transporting ATPase accessory subunit LEM3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsXu J / He Y / Wu X / Li L
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31870835 China
CitationJournal: Cell Rep / Year: 2022
Title: Conformational changes of a phosphatidylcholine flippase in lipid membranes.
Authors: Jinkun Xu / Yilin He / Xiaofei Wu / Long Li /
Abstract: Type 4 P-type ATPases (P4-ATPases) actively and selectively translocate phospholipids across membrane bilayers. Driven by ATP hydrolysis, P4-ATPases undergo conformational changes during lipid ...Type 4 P-type ATPases (P4-ATPases) actively and selectively translocate phospholipids across membrane bilayers. Driven by ATP hydrolysis, P4-ATPases undergo conformational changes during lipid flipping. It is unclear how the active flipping states of P4-ATPases are regulated in the lipid membranes, especially for phosphatidylcholine (PC)-flipping P4-ATPases whose substrate, PC, is a substantial component of membranes. Here, we report the cryoelectron microscopy structures of a yeast PC-flipping P4-ATPase, Dnf1, in lipid environments. In native yeast lipids, Dnf1 adopts a conformation in which the lipid flipping pathway is disrupted. Only when the lipid composition is changed can Dnf1 be captured in the active conformations that enable lipid flipping. These results suggest that, in the native membrane, Dnf1 may stay in an idle conformation that is unable to support the trans-membrane movement of lipids. Dnf1 may have altered conformational preferences in membranes with different lipid compositions.
History
DepositionDec 30, 2020-
Header (metadata) releaseMar 23, 2022-
Map releaseMar 23, 2022-
UpdateMar 30, 2022-
Current statusMar 30, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_30829.png

Downloads & links

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Map

FileDownload / File: emd_30829.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.055 Å
Density
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.11961678 - 0.21188985
Average (Standard dev.)0.00017492913 (±0.0033880193)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 337.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dnf1_Lem3 complex

EntireName: Dnf1_Lem3 complex
Components
  • Complex: Dnf1_Lem3 complex
    • Protein or peptide: Phospholipid-transporting ATPase DNF1
    • Protein or peptide: Alkylphosphocholine resistance protein LEM3
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE

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Supramolecule #1: Dnf1_Lem3 complex

SupramoleculeName: Dnf1_Lem3 complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)

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Macromolecule #1: Phospholipid-transporting ATPase DNF1

MacromoleculeName: Phospholipid-transporting ATPase DNF1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 178.000172 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSGTFHGDGH APMSPFEDTF QFEDNSSNED THIAPTHFDD GATSNKYSRP QVSFNDETPK NKREDAEEFT FNDDTEYDNH SFQPTPKLN NGSGTFDDVE LDNDSGEPHT NYDGMKRFRM GTKRNKKGNP IMGRSKTLKW ARKNIPNPFE DFTKDDIDPG A INRAQELR ...String:
MSGTFHGDGH APMSPFEDTF QFEDNSSNED THIAPTHFDD GATSNKYSRP QVSFNDETPK NKREDAEEFT FNDDTEYDNH SFQPTPKLN NGSGTFDDVE LDNDSGEPHT NYDGMKRFRM GTKRNKKGNP IMGRSKTLKW ARKNIPNPFE DFTKDDIDPG A INRAQELR TVYYNMPLPK DMIDEEGNPI MQYPRNKIRT TKYTPLTFLP KNILFQFHNF ANVYFLVLII LGAFQIFGVT NP GLSAVPL VVIVIITAIK DAIEDSRRTV LDLEVNNTKT HILEGVENEN VSTDNISLWR RFKKANSRLL FKFIQYCKEH LTE EGKKKR MQRKRHELRV QKTVGTSGPR SSLDSIDSYR VSADYGRPSL DYDNLEQGAG EANIVDRSLP PRTDCKFAKN YWKG VKVGD IVRIHNNDEI PADIILLSTS DTDGACYVET KNLDGETNLK VRQSLKCTNT IRTSKDIART KFWIESEGPH SNLYT YQGN MKWRNLADGE IRNEPITINN VLLRGCTLRN TKWAMGVVMF TGGDTKIMLN SGITPTKKSR ISRELNFSVV INFVLL FIL CFVSGIANGV YYDKKGRSRF SYEFGTIAGS AATNGFVSFW VAVILYQSLV PISLYISVEI IKTAQAAFIY GDVLLYN AK LDYPCTPKSW NISDDLGQVE YIFSDKTGTL TQNVMEFKKC TINGVSYGRA YTEALAGLRK RQGIDVETEG RREKAEIA K DRDTMIDELR ALSGNSQFYP EEVTFVSKEF VRDLKGASGE VQQRCCEHFM LALALCHSVL VEANPDNPKK LDLKAQSPD EAALVATARD VGFSFVGKTK KGLIIEMQGI QKEFEILNIL EFNSSRKRMS CIVKIPGLNP GDEPRALLIC KGADSIIYSR LSRQSGSNS EAILEKTALH LEQYATEGLR TLCIAQRELS WSEYEKWNEK YDIAAASLAN REDELEVVAD SIERELILLG G TAIEDRLQ DGVPDCIELL AEAGIKLWVL TGDKVETAIN IGFSCNLLNN EMELLVIKTT GDDVKEFGSE PSEIVDALLS KY LKEYFNL TGSEEEIFEA KKDHEFPKGN YAIVIDGDAL KLALYGEDIR RKFLLLCKNC RAVLCCRVSP SQKAAVVKLV KDS LDVMTL AIGDGSNDVA MIQSADVGIG IAGEEGRQAV MCSDYAIGQF RYLARLVLVH GRWSYKRLAE MIPEFFYKNM IFAL ALFWY GIYNDFDGSY LYEYTYMMFY NLAFTSLPVI FLGILDQDVN DTISLVVPQL YRVGILRKEW NQRKFLWYML DGLYQ SIIC FFFPYLVYHK NMIVTSNGLG LDHRYFVGVY VTTIAVISCN TYVLLHQYRW DWFSGLFIAL SCLVVFAWTG IWSSAI ASR EFFKAAARIY GAPSFWAVFF VAVLFCLLPR FTYDSFQKFF YPTDVEIVRE MWQHGHFDHY PPGYDPTDPN RPKVTKA GQ HGEKIIEGIA LSDNLGGSNY SRDSVVTEEI PMTFMHGEDG SPSGYQKQET WMTSPKETQD LLQSPQFQQA QTFGRGPS T NVRSSLDRTR EQMIATNQLD NRYSVERART SLDLPGVTNA ASLIGTQQNN

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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 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

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #6: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-...

MacromoleculeName: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
type: ligand / ID: 6 / Number of copies: 2 / Formula: 6PL
Molecular weightTheoretical: 763.1 Da
Chemical component information

ChemComp-6PL:
(4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / phospholipid*YM

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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

BufferpH: 7.5
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K2 BASE (4k x 4k) / Average electron dose: 8.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6roh

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 394420

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