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- PDB-7w72: Structure of a human glycosylphosphatidylinositol (GPI) transamidase -

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Basic information

Entry
Database: PDB / ID: 7w72
TitleStructure of a human glycosylphosphatidylinositol (GPI) transamidase
Components
  • (GPI transamidase component PIG- ...) x 2
  • GPI-anchor transamidase
  • Glycosylphosphatidylinositol anchor attachment 1 protein
  • Phosphatidylinositol glycan anchor biosynthesis class U protein
KeywordsMEMBRANE PROTEIN / glycosylphosphatidylinositol / transamidase
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 / Hydrolases / regulation of receptor signaling pathway via JAK-STAT / protein disulfide isomerase activity / tubulin binding ...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 / Hydrolases / regulation of receptor signaling pathway via JAK-STAT / protein disulfide isomerase activity / tubulin binding / neuron differentiation / cytoplasmic vesicle / protein-containing complex assembly / neuron apoptotic process / centrosome / endoplasmic reticulum membrane / endoplasmic reticulum / mitochondrion / proteolysis / 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 / Peptidase C13 family
Similarity search - Domain/homology
Chem-8JY / Glycosylphosphatidylinositol anchor attachment 1 protein / GPI-anchor transamidase / GPI transamidase component PIG-T / GPI transamidase component PIG-S / Phosphatidylinositol glycan anchor biosynthesis class U protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsZhang, H. / Su, J. / Li, B. / Gao, Y. / Zhang, X.C. / Zhao, Y.
Funding support China, 2items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37030304 China
Chinese Academy of SciencesXDB37030301 China
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structure of human glycosylphosphatidylinositol transamidase.
Authors: Hongwei Zhang / Jiawei Su / Bin Li / Yiwei Gao / Mengran Liu / Lingli He / Hao Xu / Yanli Dong / Xuejun Cai Zhang / Yan Zhao /
Abstract: Glycosylphosphatidylinositol (GPI) molecules are complex glycophospholipids and serve as membrane anchors for tethering many proteins to the cell surface. Attaching GPI to the protein in the ...Glycosylphosphatidylinositol (GPI) molecules are complex glycophospholipids and serve as membrane anchors for tethering many proteins to the cell surface. Attaching GPI to the protein in the endoplasmic reticulum (ER) is catalyzed by the transmembrane GPI transamidase (GPIT) complex, which is essential for maturation of the GPI-anchored proteins. The GPIT complex is known to be composed of five subunits: PIGK, PIGU, PIGT, PIGS and GPAA1. Here, we determined the structure of the human GPIT complex at a resolution of 3.1 Å using single-particle cryo-EM, elucidating its overall assembly. The PIGK subunit functions as the catalytic component, in which we identified a C206-H164-N58 triad that is critical for the transamination reaction. Transmembrane helices constitute a widely opened cleft, which is located underneath PIGK, serving as a GPI substrate-binding site. The ubiquitin E3 ligase RNF121 is visualized at the back of the complex and probably serves as a quality control factor for the GPIT complex.
History
DepositionDec 2, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
U: Phosphatidylinositol glycan anchor biosynthesis class U protein
S: GPI transamidase component PIG-S
T: GPI transamidase component PIG-T
K: GPI-anchor transamidase
A: Glycosylphosphatidylinositol anchor attachment 1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)282,62210
Polymers280,9305
Non-polymers1,6925
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27640 Å2
ΔGint-174 kcal/mol
Surface area89260 Å2

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Components

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Protein , 3 types, 3 molecules UKA

#1: Protein Phosphatidylinositol glycan anchor biosynthesis class U protein / Cell division cycle protein 91-like 1 / Protein CDC91-like 1 / GPI transamidase component PIG-U


Mass: 48448.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIGU / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9H490
#4: Protein GPI-anchor transamidase / GPI transamidase / GPI8 homolog / hGPI8 / Phosphatidylinositol-glycan biosynthesis class K protein / PIG-K


Mass: 45302.629 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIGK / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q92643, Hydrolases
#5: Protein Glycosylphosphatidylinositol anchor attachment 1 protein / GPI anchor attachment protein 1 / GAA1 protein homolog / hGAA1


Mass: 67683.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GPAA1, GAA1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: O43292

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

#2: Protein GPI transamidase component PIG-S / Phosphatidylinositol-glycan biosynthesis class S protein


Mass: 59097.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIGS / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q96S52
#3: Protein GPI transamidase component PIG-T / Phosphatidylinositol-glycan biosynthesis class T protein


Mass: 60397.691 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIGT / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q969N2

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Sugars , 2 types, 3 molecules

#6: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#8: 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 / N-Acetylglucosamine


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

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-8JY / [2-[[(2~{R})-2-hexanoyloxy-3-[(~{E})-hex-3-enoxy]propoxy]-oxidanyl-phosphoryl]oxy-3,4,5,6-tetrakis(oxidanyl)phenyl] (2~{E},4~{E})-hepta-2,4-dienoate


Mass: 622.639 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H47O13P / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca

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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: Human glycosylphosphatidylinositol transamidase / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal defocus max: 22000 nm / Nominal defocus min: 12000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61148 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00519649
ELECTRON MICROSCOPYf_angle_d0.726792
ELECTRON MICROSCOPYf_dihedral_angle_d16.8162642
ELECTRON MICROSCOPYf_chiral_restr0.0463088
ELECTRON MICROSCOPYf_plane_restr0.0053353

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