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- PDB-8k9q: Cryo-EM structure of the GPI inositol-deacylase (PGAP1/Bst1) from... -

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Basic information

Entry
Database: PDB / ID: 8k9q
TitleCryo-EM structure of the GPI inositol-deacylase (PGAP1/Bst1) from Chaetomium thermophilum
ComponentsGPI inositol-deacylase,fused thermostable green fluorescent protein
KeywordsMEMBRANE PROTEIN / Bst1 / Glycosylphosphatidylinositol / GPI anchoring / GPI-AP / GPI-AP remodelase / Integral membrane enzyme / Lipase / Lipid remodeling / Membrane enzyme / Nanodisc / PGAP1 / Transmembrane enzyme / Triad enzyme
Function / homology
Function and homology information


GPI anchor metabolic process / phosphatidylinositol deacylase activity / endoplasmic reticulum to Golgi vesicle-mediated transport / protein transport / Hydrolases; Acting on ester bonds / endoplasmic reticulum membrane
Similarity search - Function
GPI inositol-deacylase / : / GPI inositol-deacylase transmembrane domain / GPI inositol-deacylase beta-sandwich domain / GPI inositol-deacylase PGAP1-like / PGAP1-like alpha/beta domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Chem-D39 / CHOLESTEROL HEMISUCCINATE / GPI inositol-deacylase
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsHong, J. / Li, T. / Qu, Q. / Li, D.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBC
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Revision 1.2Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GPI inositol-deacylase,fused thermostable green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,1376
Polymers163,1531
Non-polymers2,9845
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 163153.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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.
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus), (gene. exp.) synthetic construct (others)
Gene: CTHT_0034210 / Production host: Homo sapiens (human)
References: UniProt: G0S652, Hydrolases; Acting on ester bonds
#2: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4
#3: Chemical ChemComp-D39 / (2~{S})-2-azanyl-3-[[(2~{R})-3-hexadecanoyloxy-2-[(~{Z})-octadec-9-enoyl]oxy-propoxy]-oxidanyl-phosphoryl]oxy-propanoic acid


Mass: 762.006 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H76NO10P
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: GPI inositol-deacylase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.163 MDa / Experimental value: NO
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
220 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot for 4.5s before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv3.3.2particle selection
4CTFFINDctffind4CTF correction
8PHENIXmodel refinement
10cryoSPARCv3.3.2initial Euler assignment
11cryoSPARCv3.3.2final Euler assignment
12cryoSPARCv3.3.2classification
13cryoSPARCv3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 407921 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027746
ELECTRON MICROSCOPYf_angle_d0.57510556
ELECTRON MICROSCOPYf_dihedral_angle_d7.3441121
ELECTRON MICROSCOPYf_chiral_restr0.0411231
ELECTRON MICROSCOPYf_plane_restr0.0051302

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