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- EMDB-35575: Cryo-EM structure of GPI-T with a chimeric GPI-anchored protein -

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Basic information

Entry
Database: EMDB / ID: EMD-35575
TitleCryo-EM structure of GPI-T with a chimeric GPI-anchored protein
Map data
Sample
  • Complex: Complex of GPI-T with a chimeric GPI-anchored protein
    • Protein or peptide: x 6 types
  • Protein or peptide: x 1 types
  • Ligand: x 12 types
Keywordscryo-EM / glycosylphosphatidylinositol / GPI / GPI anchored protein / GPI-AP / membrane protein complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


GPI-anchor transamidase activity / attachment of GPI anchor to protein / GPI-anchor transamidase complex / GPI anchor biosynthetic process / protein retention in ER lumen / Attachment of GPI anchor to uPAR / natural killer cell lectin-like receptor binding / Post-translational modification: synthesis of GPI-anchored proteins / Hydrolases / natural killer cell mediated cytotoxicity ...GPI-anchor transamidase activity / attachment of GPI anchor to protein / GPI-anchor transamidase complex / GPI anchor biosynthetic process / protein retention in ER lumen / Attachment of GPI anchor to uPAR / natural killer cell lectin-like receptor binding / Post-translational modification: synthesis of GPI-anchored proteins / Hydrolases / natural killer cell mediated cytotoxicity / natural killer cell activation / regulation of receptor signaling pathway via JAK-STAT / protein disulfide isomerase activity / tubulin binding / bioluminescence / generation of precursor metabolites and energy / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent / antigen processing and presentation of endogenous peptide antigen via MHC class Ib / neuron differentiation / positive regulation of T cell mediated cytotoxicity / cytoplasmic vesicle / protein-containing complex assembly / neuron apoptotic process / immune response / external side of plasma membrane / intracellular membrane-bounded organelle / centrosome / endoplasmic reticulum membrane / cell surface / endoplasmic reticulum / mitochondrion / proteolysis / extracellular space / extracellular region / membrane / plasma membrane / cytosol
Similarity search - Function
GPI transamidase component PIG-T / GPI transamidase component Gaa1 / GPI transamidase subunit PIG-U / Phosphatidylinositol-glycan biosynthesis class S protein / GPI-anchor transamidase / Gpi16 subunit, GPI transamidase component / Gaa1-like, GPI transamidase component / GPI transamidase subunit PIG-U / Phosphatidylinositol-glycan biosynthesis class S protein / Peptidase C13, legumain ...GPI transamidase component PIG-T / GPI transamidase component Gaa1 / GPI transamidase subunit PIG-U / Phosphatidylinositol-glycan biosynthesis class S protein / GPI-anchor transamidase / Gpi16 subunit, GPI transamidase component / Gaa1-like, GPI transamidase component / GPI transamidase subunit PIG-U / Phosphatidylinositol-glycan biosynthesis class S protein / Peptidase C13, legumain / Peptidase C13 family / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Class I Histocompatibility antigen, domains alpha 1 and 2 / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein
Similarity search - Domain/homology
Glycosylphosphatidylinositol anchor attachment 1 protein / GPI-anchor transamidase / GPI transamidase component PIG-T / GPI transamidase component PIG-S / UL16-binding protein 2 / Phosphatidylinositol glycan anchor biosynthesis class U protein / GFP-like fluorescent chromoprotein cFP484
Similarity search - Component
Biological speciesHomo sapiens (human) / Clavularia sp. (invertebrata) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsXu Y / Li T / Qu Q / Li D
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82151215 China
CitationJournal: Nat Commun / Year: 2023
Title: Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis.
Authors: Yidan Xu / Tingting Li / Zixuan Zhou / Jingjing Hong / Yulin Chao / Zhini Zhu / Ying Zhang / Qianhui Qu / Dianfan Li /
Abstract: Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a ...Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis.
History
DepositionMar 7, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35575.png

Downloads & links

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Map

FileDownload / File: emd_35575.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.932 Å
Density
Contour LevelBy AUTHOR: 0.48
Minimum - Maximum-1.8881043 - 3.0878632
Average (Standard dev.)0.0046553565 (±0.113127194)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 260.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_35575_additional_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35575_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35575_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Complex of GPI-T with a chimeric GPI-anchored protein

EntireName: Complex of GPI-T with a chimeric GPI-anchored protein
Components
  • Complex: Complex of GPI-T with a chimeric GPI-anchored protein
    • Protein or peptide: UL16-binding protein 2,GFP-like fluorescent chromoprotein cFP484
    • Protein or peptide: Glycosylphosphatidylinositol anchor attachment 1 protein,GFP-like fluorescent chromoprotein cFP484
    • Protein or peptide: GPI-anchor transamidase,GFP-like fluorescent chromoprotein cFP484
    • Protein or peptide: GPI transamidase component PIG-S,GFP-like fluorescent chromoprotein cFP484
    • Protein or peptide: GPI transamidase component PIG-T,GFP-like fluorescent chromoprotein cFP484
    • Protein or peptide: Phosphatidylinositol glycan anchor biosynthesis class U protein,GFP-like fluorescent chromoprotein cFP484
  • Protein or peptide: Met-Ser-Ser peptide
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: MAGNESIUM ION
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate
  • Ligand: Digitonin
  • Ligand: CALCIUM ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: [(2R)-1-hexadecanoyloxy-3-[[3-[[(2R)-3-hexadecanoyloxy-2-[(Z)-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-propan-2-yl] (Z)-octadec-9-enoate
  • Ligand: 2-azanylethyl [(2R,3S,4S,5S,6S)-3,4,5,6-tetrakis(oxidanyl)oxan-2-yl]methyl hydrogen phosphate
  • Ligand: 2-amino-2-deoxy-alpha-D-glucopyranose
  • Ligand: 2-azanylethyl [(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-2,4,5-tris(oxidanyl)oxan-3-yl] hydrogen phosphate
  • Ligand: [(2R)-1-[[(1S,2R,3R,4S,5S,6R)-2-hexadecanoyloxy-3,4,5,6-tetrakis(oxidanyl)cyclohexyl]oxy-oxidanyl-phosphoryl]oxy-3-octadecoxy-propan-2-yl] (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate

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Supramolecule #1: Complex of GPI-T with a chimeric GPI-anchored protein

SupramoleculeName: Complex of GPI-T with a chimeric GPI-anchored protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 469 kDa/nm

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Macromolecule #1: UL16-binding protein 2,GFP-like fluorescent chromoprotein cFP484

MacromoleculeName: UL16-binding protein 2,GFP-like fluorescent chromoprotein cFP484
type: protein_or_peptide / ID: 1
Details: A chimera proprotein containing the N- and C-terminal signal peptide of ULBP2, and a thermostable green fluorescence protein replacing the main part of ULBP2.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.573316 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAAAAATKIL LCLPLLLLLS GWSRAGGSGG SASVIKPEMK IKLRMEGAVN GHKFVIEGEG IGKPYEGTQT LDLTVEEGAP LPFSYDILT PAFQYGNRAF TKYPEDIPDY FKQAFPEGYS WERSMTYEDQ GICIATSDIT MEGDCFFYEI RFDGTNFPPN G PVMQKKTL ...String:
MAAAAATKIL LCLPLLLLLS GWSRAGGSGG SASVIKPEMK IKLRMEGAVN GHKFVIEGEG IGKPYEGTQT LDLTVEEGAP LPFSYDILT PAFQYGNRAF TKYPEDIPDY FKQAFPEGYS WERSMTYEDQ GICIATSDIT MEGDCFFYEI RFDGTNFPPN G PVMQKKTL KWEPSTEKMY VEDGVLKGDV EMALLLEGGG HYRCDFKTTY KAKKDVRLPD AHEVDHRIEI LSHDKDYNKV RL YEHAEAR YSGGGSGGGS AWSHPQFEKG GGSGGGSGGS AWSHPQFEKG SSAGAPLAMS SGTTQLRATA TTLILCCLLI ILP CFILPG I

UniProtKB: UL16-binding protein 2, GFP-like fluorescent chromoprotein cFP484, UL16-binding protein 2

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Macromolecule #2: Glycosylphosphatidylinositol anchor attachment 1 protein,GFP-like...

MacromoleculeName: Glycosylphosphatidylinositol anchor attachment 1 protein,GFP-like fluorescent chromoprotein cFP484
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clavularia sp. (invertebrata)
Molecular weightTheoretical: 96.990352 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSGLLSDPV RRRALARLVL RLNAPLCVLS YVAGIAWFLA LVFPPLTQRT YMSENAMGST MVEEQFAGGD RARAFARDFA AHRKKSGAL PVAWLERTMR SVGLEVYTQS FSRKLPFPDE THERYMVSGT NVYGILRAPR AASTESLVLT VPCGSDSTNS Q AVGLLLAL ...String:
MGSGLLSDPV RRRALARLVL RLNAPLCVLS YVAGIAWFLA LVFPPLTQRT YMSENAMGST MVEEQFAGGD RARAFARDFA AHRKKSGAL PVAWLERTMR SVGLEVYTQS FSRKLPFPDE THERYMVSGT NVYGILRAPR AASTESLVLT VPCGSDSTNS Q AVGLLLAL AAHFRGQIYW AKDIVFLVTE HDLLGTEAWL EAYHDVNVTG MQSSPLQGRA GAIQAAVALE LSSDVVTSLD VA VEGLNGQ LPNLDLLNLF QTFCQKGGLL CTLQGKLQPE DWTSLDGPLQ GLQTLLLMVL RQASGRPHGS HGLFLRYRVE ALT LRGINS FRQYKYDLVA VGKALEGMFR KLNHLLERLH QSFFLYLLPG LSRFVSIGLY MPAVGFLLLV LGLKALELWM QLHE AGMGL EEPGGAPGPS VPLPPSQGVG LASLVAPLLI SQAMGLALYV LPVLGQHVAT QHFPVAEAEA VVLTLLAIYA AGLAL PHNT HRVVSTQAPD RGWMALKLVA LIYLALQLGC IALTNFSLGF LLATTMVPTA ALAKPHGPRT LYAALLVLTS PAATLL GSL FLWRELQEAP LSLAEGWQLF LAALAQGVLE HHTYGALLFP LLSLGLYPCW LLFWNVLFWK GTLEVLFQGP GGSGGSA SV IKPEMKIKLR MEGAVNGHKF VIEGEGIGKP YEGTQTLDLT VEEGAPLPFS YDILTPAFQY GNRAFTKYPE DIPDYFKQ A FPEGYSWERS MTYEDQGICI ATSDITMEGD CFFYEIRFDG TNFPPNGPVM QKKTLKWEPS TEKMYVEDGV LKGDVEMAL LLEGGGHYRC DFKTTYKAKK DVRLPDAHEV DHRIEILSHD KDYNKVRLYE HAEARYSGGG SGGGGSGGGG DYKDDDDADY KDDDDA

UniProtKB: Glycosylphosphatidylinositol anchor attachment 1 protein, GFP-like fluorescent chromoprotein cFP484

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Macromolecule #3: GPI-anchor transamidase,GFP-like fluorescent chromoprotein cFP484

MacromoleculeName: GPI-anchor transamidase,GFP-like fluorescent chromoprotein cFP484
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases
Source (natural)Organism: Clavularia sp. (invertebrata)
Molecular weightTheoretical: 73.443062 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSAVTDSLS RAATVLATVL LLSFGSVAAS HIEDQAEQFF RSGHTNNWAV LVCTSRFWFN YRHVANTLSV YRSVKRLGIP DSHIVLMLA DDMACNPRNP KPATVFSHKN MELNVYGDDV EVDYRSYEVT VENFLRVLTG RIPPSTPRSK RLLSDDRSNI L IYMTGHGG ...String:
MGSAVTDSLS RAATVLATVL LLSFGSVAAS HIEDQAEQFF RSGHTNNWAV LVCTSRFWFN YRHVANTLSV YRSVKRLGIP DSHIVLMLA DDMACNPRNP KPATVFSHKN MELNVYGDDV EVDYRSYEVT VENFLRVLTG RIPPSTPRSK RLLSDDRSNI L IYMTGHGG NGFLKFQDSE EITNIELADA FEQMWQKRRY NELLFIIDTC QGASMYERFY SPNIMALASS QVGEDSLSHQ PD PAIGVHL MDRYTFYVLE FLEEINPASQ TNMNDLFQVC PKSLCVSTPG HRTDLFQRDP KNVLITDFFG SVRKVEITTE TIK LQQDSE IMESSYKEDQ MDEKLMEPLK YAEQLPVAQI IHQKPKLKDW HPPGGFILGL WALIIMVFFK TYGIKHMKFI FGTL EVLFQ GPGGSGGSAS VIKPEMKIKL RMEGAVNGHK FVIEGEGIGK PYEGTQTLDL TVEEGAPLPF SYDILTPAFQ YGNRA FTKY PEDIPDYFKQ AFPEGYSWER SMTYEDQGIC IATSDITMEG DCFFYEIRFD GTNFPPNGPV MQKKTLKWEP STEKMY VED GVLKGDVEMA LLLEGGGHYR CDFKTTYKAK KDVRLPDAHE VDHRIEILSH DKDYNKVRLY EHAEARYSGG GSGGGYP YD VPDYA

UniProtKB: GPI-anchor transamidase, GFP-like fluorescent chromoprotein cFP484

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Macromolecule #4: GPI transamidase component PIG-S,GFP-like fluorescent chromoprote...

MacromoleculeName: GPI transamidase component PIG-S,GFP-like fluorescent chromoprotein cFP484
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clavularia sp. (invertebrata)
Molecular weightTheoretical: 90.421062 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSAAAGAAA THLEVARGKR AALFFAAVAI VLGLPLWWKT TETYRASLPY SQISGLNALQ LRLMVPVTVV FTRESVPLDD QEKLPFTVV HEREIPLKYK MKIKCRFQKA YRRALDHEEE ALSSGSVQEA EAMLDEPQEQ AEGSLTVYVI SEHSSLLPQD M MSYIGPKR ...String:
MGSAAAGAAA THLEVARGKR AALFFAAVAI VLGLPLWWKT TETYRASLPY SQISGLNALQ LRLMVPVTVV FTRESVPLDD QEKLPFTVV HEREIPLKYK MKIKCRFQKA YRRALDHEEE ALSSGSVQEA EAMLDEPQEQ AEGSLTVYVI SEHSSLLPQD M MSYIGPKR TAVVRGIMHR EAFNIIGRRI VQVAQAMSLT EDVLAAALAD HLPEDKWSAE KRRPLKSSLG YEITFSLLNP DP KSHDVYW DIEGAVRRYV QPFLNALGAA GNFSVDSQIL YYAMLGVNPR FDSASSSYYL DMHSLPHVIN PVESRLGSSA ASL YPVLNF LLYVPELAHS PLYIQDKDGA PVATNAFHSP RWGGIMVYNV DSKTYNASVL PVRVEVDMVR VMEVFLAQLR LLFG IAQPQ LPPKCLLSGP TSEGLMTWEL DRLLWARSVE NLATATTTLT SLAQLLGKIS NIVIKDDVAS EVYKAVAAVQ KSAEE LASG HLASAFVASQ EAVTSSELAF FDPSLLHLLY FPDDQKFAIY IPLFLPMAVP ILLSLVKIFL ETRKSWRKPE KTDGTL EVL FQGPGGSGGS ASVIKPEMKI KLRMEGAVNG HKFVIEGEGI GKPYEGTQTL DLTVEEGAPL PFSYDILTPA FQYGNRA FT KYPEDIPDYF KQAFPEGYSW ERSMTYEDQG ICIATSDITM EGDCFFYEIR FDGTNFPPNG PVMQKKTLKW EPSTEKMY V EDGVLKGDVE MALLLEGGGH YRCDFKTTYK AKKDVRLPDA HEVDHRIEIL SHDKDYNKVR LYEHAEARYS GGGSGGGGG GGGGGGEQKL ISEEDL

UniProtKB: GPI transamidase component PIG-S, GFP-like fluorescent chromoprotein cFP484

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Macromolecule #5: GPI transamidase component PIG-T,GFP-like fluorescent chromoprote...

MacromoleculeName: GPI transamidase component PIG-T,GFP-like fluorescent chromoprotein cFP484
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clavularia sp. (invertebrata)
Molecular weightTheoretical: 94.20725 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSAAAMPLA LLVLLLLGPG GWCLAEPPRD SLREELVITP LPSGDVAATF QFRTRWDSEL QREGVSHYRL FPKALGQLIS KYSLRELHL SFTQGFWRTR YWGPPFLQAP SGAELWVWFQ DTVTDVDKSW KELSNVLSGI FCASLNFIDS TNTVTPTASF K PLGLANDT ...String:
MGSAAAMPLA LLVLLLLGPG GWCLAEPPRD SLREELVITP LPSGDVAATF QFRTRWDSEL QREGVSHYRL FPKALGQLIS KYSLRELHL SFTQGFWRTR YWGPPFLQAP SGAELWVWFQ DTVTDVDKSW KELSNVLSGI FCASLNFIDS TNTVTPTASF K PLGLANDT DHYFLRYAVL PREVVCTENL TPWKKLLPCS SKAGLSVLLK ADRLFHTSYH SQAVHIRPVC RNARCTSISW EL RQTLSVV FDAFITGQGK KDWSLFRMFS RTLTEPCPLA SESRVYVDIT TYNQDNETLE VHPPPTTTYQ DVILGTRKTY AIY DLLDTA MINNSRNLNI QLKWKRPPEN EAPPVPFLHA QRYVSGYGLQ KGELSTLLYN THPYRAFPVL LLDTVPWYLR LYVH TLTIT SKGKENKPSY IHYQPAQDRL QPHLLEMLIQ LPANSVTKVS IQFERALLKW TEYTPDPNHG FYVSPSVLSA LVPSM VAAK PVDWEESPLF NSLFPVSDGS NYFVRLYTEP LLVNLPTPDF SMPYNVICLT CTVVAVCYGS FYNLLTRTFH IEEPRT GGL AKRLANLIRR ARGVPPLGTL EVLFQGPGGS GGSASVIKPE MKIKLRMEGA VNGHKFVIEG EGIGKPYEGT QTLDLTV EE GAPLPFSYDI LTPAFQYGNR AFTKYPEDIP DYFKQAFPEG YSWERSMTYE DQGICIATSD ITMEGDCFFY EIRFDGTN F PPNGPVMQKK TLKWEPSTEK MYVEDGVLKG DVEMALLLEG GGHYRCDFKT TYKAKKDVRL PDAHEVDHRI EILSHDKDY NKVRLYEHAE ARYSGGGSGG GHHHHHHHHH H

UniProtKB: GPI transamidase component PIG-T, GFP-like fluorescent chromoprotein cFP484

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Macromolecule #6: Phosphatidylinositol glycan anchor biosynthesis class U protein,G...

MacromoleculeName: Phosphatidylinositol glycan anchor biosynthesis class U protein,GFP-like fluorescent chromoprotein cFP484
type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clavularia sp. (invertebrata)
Molecular weightTheoretical: 77.141695 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSAAPLVLV LVVAVTVRAA LFRSSLAEFI SERVEVVSPL SSWKRVVEGL SLLDLGVSPY SGAVFHETPL IIYLFHFLID YAELVFMIT DALTAIALYF AIQDFNKVVF KKQKLLLELD QYAPDVAELI RTPMEMRYIP LKVALFYLLN PYTILSCVAK S TCAINNTL ...String:
MGSAAPLVLV LVVAVTVRAA LFRSSLAEFI SERVEVVSPL SSWKRVVEGL SLLDLGVSPY SGAVFHETPL IIYLFHFLID YAELVFMIT DALTAIALYF AIQDFNKVVF KKQKLLLELD QYAPDVAELI RTPMEMRYIP LKVALFYLLN PYTILSCVAK S TCAINNTL IAFFILTTIK GSAFLSAIFL ALATYQSLYP LTLFVPGLLY LLQRQYIPVK MKSKAFWIFS WEYAMMYVGS LV VIICLSF FLLSSWDFIP AVYGFILSVP DLTPNIGLFW YFFAEMFEHF SLFFVCVFQI NVFFYTIPLA IKLKEHPIFF MFI QIAVIA IFKSYPTVGD VALYMAFFPV WNHLYRFLRN IFVLTCIIIV CSLLFPVLWH LWIYAGSANS NFFYAITLTF NVGQ ILLIS DYFYAFLRRE YYLTHGLYLT AKDGTEAMLV LKGTLEVLFQ GPGGSGGSAS VIKPEMKIKL RMEGAVNGHK FVIEG EGIG KPYEGTQTLD LTVEEGAPLP FSYDILTPAF QYGNRAFTKY PEDIPDYFKQ AFPEGYSWER SMTYEDQGIC IATSDI TME GDCFFYEIRF DGTNFPPNGP VMQKKTLKWE PSTEKMYVED GVLKGDVEMA LLLEGGGHYR CDFKTTYKAK KDVRLPD AH EVDHRIEILS HDKDYNKVRL YEHAEARYSG GGSGGG

UniProtKB: Phosphatidylinositol glycan anchor biosynthesis class U protein, GFP-like fluorescent chromoprotein cFP484

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Macromolecule #7: Met-Ser-Ser peptide

MacromoleculeName: Met-Ser-Ser peptide / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 323.367 Da
SequenceString:
MSS

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Macromolecule #9: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 9 / Number of copies: 13 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyl...

MacromoleculeName: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate
type: ligand / ID: 11 / Number of copies: 4 / Formula: 6OU
Molecular weightTheoretical: 717.996 Da
Chemical component information

ChemComp-6OU:
[(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate / phospholipid*YM

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Macromolecule #12: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 12 / Number of copies: 1 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM

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Macromolecule #13: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 13 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #14: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 14 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #15: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

MacromoleculeName: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / type: ligand / ID: 15 / Number of copies: 1 / Formula: LBN
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-LBN:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / phospholipid*YM

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Macromolecule #16: [(2R)-1-hexadecanoyloxy-3-[[3-[[(2R)-3-hexadecanoyloxy-2-[(Z)-oct...

MacromoleculeName: [(2R)-1-hexadecanoyloxy-3-[[3-[[(2R)-3-hexadecanoyloxy-2-[(Z)-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-propan-2-yl] (Z)-octadec-9-enoate
type: ligand / ID: 16 / Number of copies: 1 / Formula: 80T
Molecular weightTheoretical: 1.40592 KDa
Chemical component information

ChemComp-80T:
[(2R)-1-hexadecanoyloxy-3-[[3-[[(2R)-3-hexadecanoyloxy-2-[(Z)-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-propan-2-yl] (Z)-octadec-9-enoate

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Macromolecule #17: 2-azanylethyl [(2R,3S,4S,5S,6S)-3,4,5,6-tetrakis(oxidanyl)oxan-2-...

MacromoleculeName: 2-azanylethyl [(2R,3S,4S,5S,6S)-3,4,5,6-tetrakis(oxidanyl)oxan-2-yl]methyl hydrogen phosphate
type: ligand / ID: 17 / Number of copies: 2 / Formula: 80Y
Molecular weightTheoretical: 303.204 Da
Chemical component information

ChemComp-80Y:
2-azanylethyl [(2R,3S,4S,5S,6S)-3,4,5,6-tetrakis(oxidanyl)oxan-2-yl]methyl hydrogen phosphate

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Macromolecule #18: 2-amino-2-deoxy-alpha-D-glucopyranose

MacromoleculeName: 2-amino-2-deoxy-alpha-D-glucopyranose / type: ligand / ID: 18 / Number of copies: 1 / Formula: PA1
Molecular weightTheoretical: 179.171 Da
Chemical component information

ChemComp-PA1:
2-amino-2-deoxy-alpha-D-glucopyranose

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Macromolecule #19: 2-azanylethyl [(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-...

MacromoleculeName: 2-azanylethyl [(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-2,4,5-tris(oxidanyl)oxan-3-yl] hydrogen phosphate
type: ligand / ID: 19 / Number of copies: 1 / Formula: 05E
Molecular weightTheoretical: 303.204 Da
Chemical component information

ChemComp-05E:
2-azanylethyl [(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-2,4,5-tris(oxidanyl)oxan-3-yl] hydrogen phosphate

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Macromolecule #20: [(2R)-1-[[(1S,2R,3R,4S,5S,6R)-2-hexadecanoyloxy-3,4,5,6-tetrakis(...

MacromoleculeName: [(2R)-1-[[(1S,2R,3R,4S,5S,6R)-2-hexadecanoyloxy-3,4,5,6-tetrakis(oxidanyl)cyclohexyl]oxy-oxidanyl-phosphoryl]oxy-3-octadecoxy-propan-2-yl] (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate
type: ligand / ID: 20 / Number of copies: 1 / Formula: 81Q
Molecular weightTheoretical: 1.111553 KDa
Chemical component information

ChemComp-81Q:
[(2R)-1-[[(1S,2R,3R,4S,5S,6R)-2-hexadecanoyloxy-3,4,5,6-tetrakis(oxidanyl)cyclohexyl]oxy-oxidanyl-phosphoryl]oxy-3-octadecoxy-propan-2-yl] (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate

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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

Concentration8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
0.15 MNaClSodium chloride
0.1 % (w/v)DigitoninDigitonin
0.05 MTrisTris(hydroxymethyl)aminomethane

Details: 0.1 % Digitonin, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 35 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3170202
Startup modelType of model: EMDB MAP
EMDB ID:

32582

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 34261
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v4.2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v4.2.0)
FSC plot (resolution estimation)

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