+Open data
-Basic information
Entry | Database: PDB / ID: 7zay | ||||||||||||
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Title | Human heparan sulfate polymerase complex EXT1-EXT2 | ||||||||||||
Components |
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Keywords | TRANSFERASE / Heparan sulfate biosynthesis / Enzyme complex / Glycosyltransferase / Golgi | ||||||||||||
Function / homology | Function and homology information glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / hypersensitivity / heart field specification / lymphocyte adhesion to endothelial cell of high endothelial venule / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / smoothened signaling pathway involved in lung development / developmental growth involved in morphogenesis ...glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / hypersensitivity / heart field specification / lymphocyte adhesion to endothelial cell of high endothelial venule / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / smoothened signaling pathway involved in lung development / developmental growth involved in morphogenesis / sweat gland development / perichondral bone morphogenesis / mesenchymal cell differentiation involved in bone development / response to leukemia inhibitory factor / UDP-N-acetylglucosamine transferase complex / chondroitin sulfate metabolic process / response to heparin / chondrocyte hypertrophy / embryonic skeletal joint development / hematopoietic stem cell migration to bone marrow / heparan sulfate proteoglycan biosynthetic process, polysaccharide chain biosynthetic process / fluid transport / glucuronosyltransferase activity / limb joint morphogenesis / tight junction organization / heparin biosynthetic process / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / stomach development / sebaceous gland development / glomerular basement membrane development / heparan sulfate proteoglycan biosynthetic process / HS-GAG biosynthesis / glycosaminoglycan biosynthetic process / lymphocyte migration into lymphoid organs / dendrite self-avoidance / hematopoietic stem cell homeostasis / chondrocyte proliferation / glandular epithelial cell differentiation / dendritic cell migration / endochondral bone morphogenesis / sulfation / endochondral bone growth / podocyte differentiation / sodium ion homeostasis / acetylglucosaminyltransferase activity / basement membrane organization / cartilage development involved in endochondral bone morphogenesis / polysaccharide biosynthetic process / cranial skeletal system development / vocalization behavior / stem cell division / olfactory bulb development / vacuole organization / multicellular organismal-level water homeostasis / leukocyte tethering or rolling / response to light intensity / endoderm development / endochondral ossification / fear response / regulation of tumor necrosis factor-mediated signaling pathway / protein N-linked glycosylation / cellular response to fibroblast growth factor stimulus / collagen fibril organization / neural crest cell differentiation / ossification involved in bone maturation / motor behavior / cell adhesion mediated by integrin / optic nerve development / hair follicle morphogenesis / epithelial tube branching involved in lung morphogenesis / cell fate commitment / heart contraction / BMP signaling pathway / glycosyltransferase activity / protein glycosylation / mesoderm development / antigen processing and presentation / social behavior / mesoderm formation / catalytic complex / hematopoietic stem cell differentiation / canonical Wnt signaling pathway / blood vessel remodeling / fibroblast growth factor receptor signaling pathway / chondrocyte differentiation / bone resorption / ossification / gastrulation / synaptic transmission, glutamatergic / axon guidance / regulation of blood pressure / wound healing / protein catabolic process / vasodilation / multicellular organism growth / cellular response to virus / gene expression / protein-containing complex assembly / protein heterodimerization activity Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å | ||||||||||||
Authors | Leisico, F. / Omeiri, J. / Hons, M. / Schoehn, G. / Lortat-Jacob, H. / Wild, R. | ||||||||||||
Funding support | France, 3items
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Citation | Journal: Nat Commun / Year: 2022 Title: Structure of the human heparan sulfate polymerase complex EXT1-EXT2. Authors: Francisco Leisico / Juneina Omeiri / Christine Le Narvor / Joël Beaudouin / Michael Hons / Daphna Fenel / Guy Schoehn / Yohann Couté / David Bonnaffé / Rabia Sadir / Hugues Lortat-Jacob / Rebekka Wild / Abstract: Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi- ...Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7zay.cif.gz | 245.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7zay.ent.gz | 191.8 KB | Display | PDB format |
PDBx/mmJSON format | 7zay.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7zay_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7zay_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7zay_validation.xml.gz | 50.4 KB | Display | |
Data in CIF | 7zay_validation.cif.gz | 74.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/za/7zay ftp://data.pdbj.org/pub/pdb/validation_reports/za/7zay | HTTPS FTP |
-Related structure data
Related structure data | 14582MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 85261.859 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: EXT1 / Cell line (production host): Freestyle 293-F / Production host: Homo sapiens (human) References: UniProt: Q16394, glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase, N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase |
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#2: Protein | Mass: 79056.836 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: EXT2 / Cell line (production host): Freestyle 293-F / Production host: Homo sapiens (human) References: UniProt: Q93063, glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase, N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase |
#3: 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 Source method: isolated from a genetically manipulated source |
#4: Sugar | ChemComp-NAG / |
#5: Chemical | ChemComp-UDP / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Hetero-dimeric complex EXT1-EXT2 / Type: COMPLEX Details: local resolution-filtered map generated using a B-factor of -30 A2 Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.16 MDa / Experimental value: YES |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Homo sapiens (human) / Cell: Freestyle 293-F |
Buffer solution | pH: 7.2 |
Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Purified EXT1-EXT2 complex at 0.4mg/mL was mixed with 1mM of UDP-GlcNAc, 1mM UDP-GlcA and 1mM MnCl2 and incubated 15 min on ice before applying 4 uL of the sample onto a glow discharged ...Details: Purified EXT1-EXT2 complex at 0.4mg/mL was mixed with 1mM of UDP-GlcNAc, 1mM UDP-GlcA and 1mM MnCl2 and incubated 15 min on ice before applying 4 uL of the sample onto a glow discharged Quantifoil holey carbon grid (R1.2/1.3, 300 mesh, copper). |
Specimen support | Grid material: COPPER / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 46 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 286390 / Symmetry type: POINT | ||||||||||||||||||||||||
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