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- PDB-8k9t: Cryo-EM structure of the products-bound PGAP1(Bst1)-S327A from Ch... -

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Basic information

Entry
Database: PDB / ID: 8k9t
TitleCryo-EM structure of the products-bound PGAP1(Bst1)-S327A from Chaetonium thermophilum
Components
  • GPI inositol-deacylase,MCherry protein
  • Green fluorescent protein,Complement decay-accelerating factor
KeywordsMEMBRANE PROTEIN / Bst1 / Glycosylphosphatidylinositol / GPI anchoring / GPI-AP / GPI-AP remodelase / Integral membrane enzyme / Lipase / Lipid remodeling / Membrane enzyme / Nanodisc / PGAP1 / TGP / Thermostable green fluorescence protein / Transmembrane enzyme / Triad enzyme.
Function / homology
Function and homology information


GPI anchor metabolic process / phosphatidylinositol deacylase activity / regulation of lipopolysaccharide-mediated signaling pathway / negative regulation of complement activation / negative regulation of complement activation, classical pathway / regulation of complement-dependent cytotoxicity / regulation of complement activation / T cell mediated immunity / respiratory burst / positive regulation of CD4-positive, alpha-beta T cell activation ...GPI anchor metabolic process / phosphatidylinositol deacylase activity / regulation of lipopolysaccharide-mediated signaling pathway / negative regulation of complement activation / negative regulation of complement activation, classical pathway / regulation of complement-dependent cytotoxicity / regulation of complement activation / T cell mediated immunity / respiratory burst / positive regulation of CD4-positive, alpha-beta T cell activation / positive regulation of CD4-positive, alpha-beta T cell proliferation / Class B/2 (Secretin family receptors) / ficolin-1-rich granule membrane / endoplasmic reticulum to Golgi vesicle-mediated transport / side of membrane / COPI-mediated anterograde transport / transport vesicle / endoplasmic reticulum-Golgi intermediate compartment membrane / bioluminescence / secretory granule membrane / generation of precursor metabolites and energy / complement activation, classical pathway / Regulation of Complement cascade / positive regulation of T cell cytokine production / protein transport / virus receptor activity / positive regulation of cytosolic calcium ion concentration / Hydrolases; Acting on ester bonds / membrane raft / Golgi membrane / innate immune response / lipid binding / Neutrophil degranulation / endoplasmic reticulum membrane / cell surface / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
GPI inositol-deacylase PGAP1-like / GPI inositol-deacylase / PGAP1-like protein / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related ...GPI inositol-deacylase PGAP1-like / GPI inositol-deacylase / PGAP1-like protein / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Chem-05E / Chem-80Y / CHOLESTEROL / Chem-LYI / alpha-D-mannopyranose / 2-amino-2-deoxy-alpha-D-glucopyranose / PALMITIC ACID / Uncharacterized protein / GPI inositol-deacylase / Complement decay-accelerating factor
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
Psychromonas sp. B3M02 (bacteria)
synthetic construct (others)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsLi, T. / Hong, J. / 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
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GPI inositol-deacylase,MCherry protein
B: Green fluorescent protein,Complement decay-accelerating factor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,28811
Polymers191,2402
Non-polymers3,0499
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein GPI inositol-deacylase,MCherry protein


Mass: 161345.547 Da / Num. of mol.: 1 / Mutation: S327A,W148S,I166V,Q168Y,I202R
Source method: isolated from a genetically manipulated source
Details: Residues 1-1186 is GPI inositol-deacylase (Uniprot ID G0S652) with a S327A mutation. Residues 1187-1200 is the linker with a 3 C protease digestion site. Residues 1201-1435 is a fused ...Details: Residues 1-1186 is GPI inositol-deacylase (Uniprot ID G0S652) with a S327A mutation. Residues 1187-1200 is the linker with a 3 C protease digestion site. Residues 1201-1435 is a fused mCherry protein (Uniprot ID A0A366VY15 with the following four mutations that extend its fluorescence lifetime: W148S, I166V, Q168Y and I202R). Residues 1436-1447 is the linker with a His tag.
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus), (gene. exp.) Psychromonas sp. B3M02 (bacteria)
Gene: CTHT_0034210, DS885_16260 / Production host: Homo sapiens (human)
References: UniProt: G0S652, UniProt: A0A366VY15, Hydrolases; Acting on ester bonds
#2: Protein Green fluorescent protein,Complement decay-accelerating factor


Mass: 29894.154 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 1-6 is a linker. Residues 7-231 is a thermostable green fluorescent protein (PDB entry 4TZA, residue 5-229). Residues 232-263 is a linker with an expression tag. Residues 264-272 is ...Details: Residues 1-6 is a linker. Residues 7-231 is a thermostable green fluorescent protein (PDB entry 4TZA, residue 5-229). Residues 232-263 is a linker with an expression tag. Residues 264-272 is human CD55 (Uniprot ID P08174, residue P345-S353)
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Homo sapiens (human)
Gene: CD55, CR, DAF / Production host: Homo sapiens (human) / References: UniProt: P08174

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Sugars , 2 types, 3 molecules

#4: Sugar ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
#6: Sugar ChemComp-PA1 / 2-amino-2-deoxy-alpha-D-glucopyranose / alpah-D-glucosamine / 2-amino-2-deoxy-alpha-D-glucose / 2-amino-2-deoxy-D-glucose / 2-amino-2-deoxy-glucose


Type: D-saccharide, alpha linking / Mass: 179.171 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO5 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-glucopyranosamineCOMMON NAMEGMML 1.0
a-D-GlcpNIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 6 molecules

#3: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2
#5: Chemical 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


Mass: 303.204 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18NO9P / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-LYI / [(2~{R})-1-octadecoxy-3-[oxidanyl-[(2~{R},3~{R},5~{S},6~{R})-2,3,4,5,6-pentakis(oxidanyl)cyclohexyl]oxy-phosphoryl]oxy-propan-2-yl] (5~{Z},8~{Z},11~{Z},14~{Z})-icosa-5,8,11,14-tetraenoate


Mass: 873.144 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H85O12P / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#9: Chemical ChemComp-80Y / 2-azanylethyl [(2R,3S,4S,5S,6S)-3,4,5,6-tetrakis(oxidanyl)oxan-2-yl]methyl hydrogen phosphate


Mass: 303.204 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18NO9P / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Complex of PGAP1 with a chimera GPI-anchored protein / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.194 MDa / Experimental value: NO
Source (natural)Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (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
30.008 %glyco-diosgeninGDN1
SpecimenConc.: 14.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 353585 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047959
ELECTRON MICROSCOPYf_angle_d0.58310874
ELECTRON MICROSCOPYf_dihedral_angle_d7.4881190
ELECTRON MICROSCOPYf_chiral_restr0.0441287
ELECTRON MICROSCOPYf_plane_restr0.0061344

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