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- PDB-7wld: Cryo-EM structure of the human glycosylphosphatidylinositol trans... -

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Basic information

Entry
Database: PDB / ID: 7wld
TitleCryo-EM structure of the human glycosylphosphatidylinositol transamidase complex at 2.53 Angstrom resolution
Components
  • (GPI transamidase component PIG- ...) x 2
  • GPI-anchor transamidase
  • Glycosylphosphatidylinositol anchor attachment 1 protein
  • Phosphatidylinositol glycan anchor biosynthesis class U protein
KeywordsTRANSFERASE / GPI anchoring / GPI-AP / glycosylphosphatidylinositol transamidase / GPI transamidase / membrane protein complex
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-05E / Chem-06O / Chem-BJR / CARDIOLIPIN / CHOLESTEROL / Chem-DKB / Chem-P5S / 2-amino-2-deoxy-alpha-D-glucopyranose / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / CHOLESTEROL HEMISUCCINATE ...Chem-05E / Chem-06O / Chem-BJR / CARDIOLIPIN / CHOLESTEROL / Chem-DKB / Chem-P5S / 2-amino-2-deoxy-alpha-D-glucopyranose / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / CHOLESTEROL HEMISUCCINATE / 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: 2.53 Å
AuthorsXu, Y. / Li, T. / Luo, Y. / Chao, Y. / Jia, G. / Zhou, Z. / Su, Z. / Qu, Q. / Li, D.
Funding support China, 5items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82151215 China
National Natural Science Foundation of China (NSFC)31870726 China
National Natural Science Foundation of China (NSFC)82041016 China
National Natural Science Foundation of China (NSFC)32070049 China
Ministry of Science and Technology (MoST, China)2021YFA1301900 China
CitationJournal: Nat Commun / Year: 2022
Title: Molecular insights into biogenesis of glycosylphosphatidylinositol anchor proteins.
Authors: Yidan Xu / Guowen Jia / Tingting Li / Zixuan Zhou / Yitian Luo / Yulin Chao / Juan Bao / Zhaoming Su / Qianhui Qu / Dianfan Li /
Abstract: Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. The removal of a ...Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. The removal of a hydrophobic signal peptide and covalent attachment of GPI at the new carboxyl terminus are catalyzed by an endoplasmic reticulum membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GPI-T at a global 2.53-Å resolution, revealing an equimolar heteropentameric assembly. Structure-based mutagenesis suggests a legumain-like mechanism for the recognition and cleavage of proprotein substrates, and an endogenous GPI in the structure defines a composite cavity for the lipid substrate. This elongated active site, stemming from the membrane and spanning an additional ~22-Å space toward the catalytic dyad, is structurally suited for both substrates which feature an amphipathic pattern that matches this geometry. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.
History
DepositionJan 13, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 27, 2022Provider: repository / Type: Initial release
Revision 1.1May 25, 2022Group: 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.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jun 1, 2022Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)451,00533
Polymers435,7535
Non-polymers15,25228
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39840 Å2
ΔGint-270 kcal/mol
Surface area97730 Å2

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Components

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Protein , 3 types, 3 molecules GKU

#1: Protein Glycosylphosphatidylinositol anchor attachment 1 protein / 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: GPAA1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: O43292
#2: Protein GPI-anchor transamidase / GPI transamidase / GPI8 homolog / hGPI8 / Phosphatidylinositol-glycan biosynthesis class K protein / PIG-K


Mass: 73443.062 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 Phosphatidylinositol glycan anchor biosynthesis class U protein / 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: PIGU / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9H490

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

#3: Protein GPI transamidase component PIG-S / 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: PIGS / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q96S52
#4: Protein GPI transamidase component PIG-T / Phosphatidylinositol-glycan biosynthesis class T protein


Mass: 94207.250 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 , 3 types, 4 molecules

#6: 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: obtained synthetically
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
#13: 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
#16: 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 , 11 types, 24 molecules

#7: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C31H50O4
#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-BJR / (4S,7R)-7-[(hexadecanoyloxy)methyl]-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-3,5,8-trioxa-4lambda~5~-phosphahexacosan-1-aminium


Mass: 763.100 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H85NO8P
#10: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#11: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#12: Chemical ChemComp-P5S / O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine / phosphatidyl serine


Mass: 792.075 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO10P
#14: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C27H46O
#15: 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
#17: Chemical ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C81H156O17P2 / Comment: phospholipid*YM
#18: Chemical ChemComp-DKB / [(2R)-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] octadecanoate


Mass: 720.012 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H78NO8P
#19: Chemical ChemComp-06O / [(2~{R})-1-hexadecanoyloxy-3-[[(1~{S},2~{R},3~{R},4~{S},5~{S},6~{R})-2-hexadecanoyloxy-3,4,5,6-tetrakis(oxidanyl)cyclohexyl]oxy-oxidanyl-phosphoryl]oxy-propan-2-yl] (9~{Z},11~{Z})-octadeca-9,11-dienoate


Mass: 1073.462 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C59H109O14P / 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: GPI transamidase complex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTris(hydroxymethyl)aminomethane hydrochlorideTris-HCl1
30.1 %Digitonin1
SpecimenConc.: 20 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 %

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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: 53 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4CTF correction
9cryoSPARC3.1initial Euler assignment
10cryoSPARC3.1final Euler assignment
11cryoSPARC3.1classification
12cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2959791
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151590 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 2.53 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320291
ELECTRON MICROSCOPYf_angle_d0.65627724
ELECTRON MICROSCOPYf_dihedral_angle_d9.033095
ELECTRON MICROSCOPYf_chiral_restr0.0433251
ELECTRON MICROSCOPYf_plane_restr0.0053328

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