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- EMDB-36995: Cryo-EM structure of the GPI inositol-deacylase (PGAP1/Bst1) from... -

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Basic information

Entry
Database: EMDB / ID: EMD-36995
TitleCryo-EM structure of the GPI inositol-deacylase (PGAP1/Bst1) from Chaetomium thermophilum
Map data
Sample
  • Complex: GPI inositol-deacylase
    • Protein or peptide: GPI inositol-deacylase,fused thermostable green fluorescent protein
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: (2~{S})-2-azanyl-3-[[(2~{R})-3-hexadecanoyloxy-2-[(~{Z})-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-propanoic acid
KeywordsBst1 / Glycosylphosphatidylinositol / GPI anchoring / GPI-AP / GPI-AP remodelase / Integral membrane enzyme / Lipase / Lipid remodeling / Membrane enzyme / Membrane protein / Nanodisc / PGAP1 / Transmembrane enzyme / Triad enzyme
Function / homologyGPI inositol-deacylase / GPI inositol-deacylase PGAP1-like / PGAP1-like protein / hydrolase activity, acting on ester bonds / protein transport / Alpha/Beta hydrolase fold / Hydrolases; Acting on ester bonds / endoplasmic reticulum membrane / GPI inositol-deacylase
Function and homology information
Biological speciesThermochaetoides thermophila (fungus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsHong J / 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: 2024
Title: Molecular basis of the inositol deacylase PGAP1 involved in quality control of GPI-AP biogenesis.
Authors: Jingjing Hong / Tingting Li / Yulin Chao / Yidan Xu / Zhini Zhu / Zixuan Zhou / Weijie Gu / Qianhui Qu / Dianfan Li /
Abstract: The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates ...The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates the inositol in nascent GPI-APs. Impairment of PGAP1 activity leads to developmental diseases in humans and fatality and infertility in animals. Here, we present three PGAP1 structures (2.66-2.84 Å), revealing its 10-transmembrane architecture and product-enzyme interaction details. PGAP1 holds GPI-AP acyl chains in an optimally organized, guitar-shaped cavity with apparent energetic penalties from hydrophobic-hydrophilic mismatches. However, abundant glycan-mediated interactions in the lumen counterbalance these repulsions, likely conferring substrate fidelity and preventing off-target hydrolysis of bulk membrane lipids. Structural and biochemical analyses uncover a serine hydrolase-type catalysis with atypical features and imply mechanisms for substrate entrance and product release involving a drawing compass movement of GPI-APs. Our findings advance the mechanistic understanding of GPI-AP remodeling.
History
DepositionAug 1, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36995.png

Downloads & links

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Map

FileDownload / File: emd_36995.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 280 pix.
= 232.96 Å		0.83 Å/pix.
x 280 pix.
= 232.96 Å		0.83 Å/pix.
x 280 pix.
= 232.96 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.743
Minimum - Maximum-2.9297326 - 4.7250338
Average (Standard dev.)0.0010319774 (±0.17257603)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 232.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36995_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: #1

Fileemd_36995_half_map_2.map
Projections & Slices
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Sample components

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Entire : GPI inositol-deacylase

EntireName: GPI inositol-deacylase
Components
  • Complex: GPI inositol-deacylase
    • Protein or peptide: GPI inositol-deacylase,fused thermostable green fluorescent protein
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: (2~{S})-2-azanyl-3-[[(2~{R})-3-hexadecanoyloxy-2-[(~{Z})-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-propanoic acid

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Supramolecule #1: GPI inositol-deacylase

SupramoleculeName: GPI inositol-deacylase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 163 KDa

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Macromolecule #1: GPI inositol-deacylase,fused thermostable green fluorescent protein

MacromoleculeName: GPI inositol-deacylase,fused thermostable green fluorescent protein
type: protein_or_peptide / ID: 1
Details: The protein is a fusion protein with expression tag. Residues 1187-1196 is the linker with a 3 C protease digestion site. Residues 1197-1427 is a fused thermostable green fluorescent protein ...Details: The protein is a fusion protein with expression tag. Residues 1187-1196 is the linker with a 3 C protease digestion site. Residues 1197-1427 is a fused thermostable green fluorescent protein (PDB entry 4TZA, residue 5-229). Residues 1428-1469 is the linker a Strep II tag and a His tag.
Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 163.153094 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSRSLSSAS SDDDDAPPIR VPRVNQCATS RTKDSQSPAQ SASKLDRRRS ADRRPSFSAN RRSGTGAGTG TGTGIANWRP FDSRDATVE RAGSSTATTA TTPPPSSSLG LMLAANGAVQ EKEMVMMGKA QEHGFVGRRA PWRSPWAISV FAFVTSLLGI G LLLAVIHS ...String:
MGSRSLSSAS SDDDDAPPIR VPRVNQCATS RTKDSQSPAQ SASKLDRRRS ADRRPSFSAN RRSGTGAGTG TGTGIANWRP FDSRDATVE RAGSSTATTA TTPPPSSSLG LMLAANGAVQ EKEMVMMGKA QEHGFVGRRA PWRSPWAISV FAFVTSLLGI G LLLAVIHS SVTRQIDPKG CRMSYMRPSY AKLSDFDTEH TRLASKYSLY LYREQGIDHD VKVRGVPVLF IPGNAGSYKQ VR PIAAEAA NYFHDVLQHD EAALRAGVRS LDFFTVDFNE DITAFHGQTL LDQAEYLNEA IRYILSLYLD PRVSERDPDL PDP TSVIVL GHSMGGIVAR TMLIMPNYQH NSINTIITMS APHARPPVSF DGQIVQTYKD INNYWRHAYS QKWANDNPLW HVTL VSIAG GGLDTVVPSD YASIESLVPD THGFTVFTST IPNVWTSMDH QAILWCDQFR KVIIRALFDI VDVHRASQTK PRAQR MRVF KKWFLSGMET VAEKIAPTSD PTTLLIVDDK SDSITAEGER LVLRELGTQG SVRAHLMPIP PPGSPELKRF TLLTDT KLD KPGENGKLEV MFCSVIPSQP NPTGPAIPSQ LDLSKGNAGT TRLACTNVAP DVITLPASTR FARFPFSVRK EAEIPPF SY LEYVLDDISE HQFVAVIEKA TIPTPGFVIA EFSDHSNSHH TRHIGLRNLL TFGISLRLPS NRPMMSEVRI PSVKSSLL A YNLRISALEC SGRKDLFAPL VRQYLAEPYE SKYFVNARQA AVSLHGVAPY VPPPMSREPE AEGLAFQLWT DPTCNSSIQ VDLTVDVMGS LGKLYMRYRT VFAAFPLFIV SLVLRKQFQV YDSTGSFITF AEGLDLSLRQ SIPVMLIVLA ALTLSTTKMA PSSSAGLWH WGGNTTFTNF HQNDLLIGTQ DPFFLFLIPL IGIICVGVCT VVNYIALSLT RLISVVISFI GFLTVRFGWV N AEDRRRPS NPAIFPPSSP RRRMITTAVL LFLVSTMIPY QLAYLVACLV QLGTLVRAQR ISSELRSPAN SNFHNYVHSI FI LMLWILP INLPTLVVWM HNLSVHWLTP FTSHHNVFSI MPFILLVETH TTGQMIPRTG GTGNGRCCVL LRHITSILLL SLA LYAAVY GVSYAYTLHQ FVNLFAFWLV MVHSTADDWS LTGLRQLILH NRNNANNKSE TGSRKRGKEP GTLEVLFQGP GGSG GSASV IKPEMKIKLR MEGAVNGHKF VIEGEGIGKP YEGTQTLDLT VEEGAPLPFS YDILTPAFQY GNRAFTKYPE DIPDY FKQA FPEGYSWERS MTYEDQGICI ATSDITMEGD CFFYEIRFDG TNFPPNGPVM QKKTLKWEPS TEKMYVEDGV LKGDVE MAL LLEGGGHYRC DFKTTYKAKK DVRLPDAHEV DHRIEILSHD KDYNKVRLYE HAEARYSGGG SGGGSAWSHP QFEKGGG SG GGSGGSAWSH PQFEKGSHHH HHHHHHH

UniProtKB: GPI inositol-deacylase

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Macromolecule #2: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #3: (2~{S})-2-azanyl-3-[[(2~{R})-3-hexadecanoyloxy-2-[(~{Z})-octadec-...

MacromoleculeName: (2~{S})-2-azanyl-3-[[(2~{R})-3-hexadecanoyloxy-2-[(~{Z})-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-propanoic acid
type: ligand / ID: 3 / Number of copies: 2 / Formula: D39
Molecular weightTheoretical: 762.006 Da
Chemical component information

ChemComp-D39:
(2~{S})-2-azanyl-3-[[(2~{R})-3-hexadecanoyloxy-2-[(~{Z})-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-propanoic acid

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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

Concentration7.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4.5s before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.2) / Number images used: 407921
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.3.2)

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