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- PDB-8imy: Cryo-EM structure of GPI-T (inactive mutant) with GPI and proULBP... -

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Basic information

Entry
Database: PDB / ID: 8imy
TitleCryo-EM structure of GPI-T (inactive mutant) with GPI and proULBP2, a proprotein substrate
Components
  • (GPI transamidase component PIG- ...) x 2
  • GPI-anchor transamidase,GFP-like fluorescent chromoprotein cFP484
  • Glycosylphosphatidylinositol anchor attachment 1 protein,GFP-like fluorescent chromoprotein cFP484
  • Phosphatidylinositol glycan anchor biosynthesis class U protein,GFP-like fluorescent chromoprotein cFP484
  • UL16-binding protein 2
KeywordsMEMBRANE PROTEIN / cryo-EM / glycosylphosphatidylinositol / GPI / GPI anchored protein / GPI-AP / membrane protein complex / proprotein
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 activation ...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 activation / regulation of receptor signaling pathway via JAK-STAT / natural killer cell mediated cytotoxicity / protein disulfide isomerase activity / tubulin binding / bioluminescence / antigen processing and presentation of endogenous peptide antigen via MHC class Ib / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent / generation of precursor metabolites and energy / neuron differentiation / positive regulation of T cell mediated cytotoxicity / cytoplasmic vesicle / protein-containing complex assembly / neuron apoptotic process / receptor ligand activity / 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
Chem-05E / Chem-6OU / Chem-80T / Chem-80Y / Chem-81Q / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / 2-amino-2-deoxy-alpha-D-glucopyranose / CHOLESTEROL HEMISUCCINATE / Glycosylphosphatidylinositol anchor attachment 1 protein / GPI-anchor transamidase ...Chem-05E / Chem-6OU / Chem-80T / Chem-80Y / Chem-81Q / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / 2-amino-2-deoxy-alpha-D-glucopyranose / CHOLESTEROL HEMISUCCINATE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsLi, T. / Xu, Y. / Qu, Q. / Li, D.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: 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
Revision 1.2Oct 23, 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

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Assembly

Deposited unit
G: Glycosylphosphatidylinositol anchor attachment 1 protein,GFP-like fluorescent chromoprotein cFP484
K: GPI-anchor transamidase,GFP-like fluorescent chromoprotein cFP484
U: Phosphatidylinositol glycan anchor biosynthesis class U protein,GFP-like fluorescent chromoprotein cFP484
T: GPI transamidase component PIG-T,GFP-like fluorescent chromoprotein cFP484
S: GPI transamidase component PIG-S,GFP-like fluorescent chromoprotein cFP484
D: UL16-binding protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)483,22944
Polymers463,1466
Non-polymers20,08338
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 4 molecules GKUD

#1: Protein Glycosylphosphatidylinositol anchor attachment 1 protein,GFP-like fluorescent chromoprotein cFP484 / GPI anchor attachment protein 1 / GAA1 protein homolog / hGAA1


Mass: 96990.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Clavularia sp. (invertebrata)
Gene: GPAA1, GAA1 / Production host: Homo sapiens (human) / References: UniProt: O43292, UniProt: Q9U6Y3
#2: Protein GPI-anchor transamidase,GFP-like fluorescent chromoprotein cFP484 / GPI transamidase / GPI8 homolog / hGPI8 / Phosphatidylinositol-glycan biosynthesis class K protein / PIG-K


Mass: 73426.992 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Clavularia sp. (invertebrata)
Gene: PIGK, GPI8 / Production host: Homo sapiens (human) / References: UniProt: Q92643, UniProt: Q9U6Y3, Hydrolases
#3: Protein Phosphatidylinositol glycan anchor biosynthesis class U protein,GFP-like fluorescent chromoprotein cFP484 / Cell division cycle protein 91-like 1 / Protein CDC91-like 1 / GPI transamidase component PIG-U


Mass: 80691.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Clavularia sp. (invertebrata)
Gene: PIGU, CDC91L1, PSEC0205, UNQ3055/PRO9875 / Production host: Homo sapiens (human) / References: UniProt: Q9H490, UniProt: Q9U6Y3
#6: Protein UL16-binding protein 2 / ALCAN-alpha / NKG2D ligand 2 / N2DL-2 / NKG2DL2 / Retinoic acid early transcript 1H


Mass: 28790.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ULBP2, N2DL2, RAET1H, UNQ463/PRO791 / Production host: Homo sapiens (human) / References: UniProt: Q9BZM5

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GPI transamidase component PIG- ... , 2 types, 2 molecules TS

#4: Protein GPI transamidase component PIG-T,GFP-like fluorescent chromoprotein cFP484 / Phosphatidylinositol-glycan biosynthesis class T protein


Mass: 92825.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Clavularia sp. (invertebrata)
Gene: PIGT, CGI-06, PSEC0163, UNQ716/PRO1379 / Production host: Homo sapiens (human) / References: UniProt: Q969N2, UniProt: Q9U6Y3
#5: Protein GPI transamidase component PIG-S,GFP-like fluorescent chromoprotein cFP484 / Phosphatidylinositol-glycan biosynthesis class S protein


Mass: 90421.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Clavularia sp. (invertebrata)
Gene: PIGS, UNQ1873/PRO4316 / Production host: Homo sapiens (human) / References: UniProt: Q96S52, UniProt: Q9U6Y3

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Sugars , 3 types, 4 molecules

#7: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#16: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#18: 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 , 10 types, 34 molecules

#8: Chemical ChemComp-AJP / Digitonin


Mass: 1229.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM
#9: Chemical
ChemComp-6OU / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate


Mass: 717.996 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C39H76NO8P / Comment: phospholipid*YM
#10: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 19 / Source method: obtained synthetically / Formula: C31H50O4
#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#12: 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
#13: 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.: 2 / Source method: obtained synthetically / Formula: C8H18NO9P / Feature type: SUBJECT OF INVESTIGATION
#14: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#15: Chemical ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#17: Chemical 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


Mass: 1111.553 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C63H115O13P / Feature type: SUBJECT OF INVESTIGATION
#19: Chemical 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


Mass: 1405.920 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C77H146O17P2

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Details

Has ligand of interestY
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: Complex of GPI-T (inactive mutant) with GPI and proULBP2
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 463 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Details: 0.1 % Digitonin, 150 mM NaCl, 20 mM Tris-HCl pH 8.0
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 Msodium chlorideNaCl1
20.1 %DigitoninDigitonin1
30.02 MTris (hydroxymethyl) aminomethaneTris1
SpecimenConc.: 25 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 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 magnification: 105000 X / Calibrated magnification: 53648 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4555

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
3SerialEM3.8.0image acquisition
5CTFFIND4CTF correction
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2classification
12cryoSPARC3.3.23D reconstruction
Image processingDetails: The images were high-pass filtered and normalized
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2981565
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176889 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00620553
ELECTRON MICROSCOPYf_angle_d0.66428089
ELECTRON MICROSCOPYf_dihedral_angle_d16.5363581
ELECTRON MICROSCOPYf_chiral_restr0.0453318
ELECTRON MICROSCOPYf_plane_restr0.0063370

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