+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14582 | ||||||||||||
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Title | Human heparan sulfate polymerase complex EXT1-EXT2 | ||||||||||||
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Sample |
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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 / hematopoietic stem cell homeostasis / chondrocyte proliferation / glandular epithelial cell differentiation / dendritic cell migration / endochondral bone morphogenesis / sulfation / endochondral bone growth / podocyte differentiation / acetylglucosaminyltransferase activity / sodium ion homeostasis / dendrite self-avoidance / response to light intensity / 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 / leukocyte tethering or rolling / multicellular organismal-level water homeostasis / endoderm development / endochondral ossification / fear response / regulation of tumor necrosis factor-mediated signaling pathway / cellular response to fibroblast growth factor stimulus / protein N-linked glycosylation / collagen fibril organization / neural crest cell differentiation / ossification involved in bone maturation / motor behavior / optic nerve development / cell adhesion mediated by integrin / epithelial tube branching involved in lung morphogenesis / hair follicle morphogenesis / heart contraction / glycosyltransferase activity / protein glycosylation / antigen processing and presentation / mesoderm development / social behavior / mesoderm formation / catalytic complex / hematopoietic stem cell differentiation / blood vessel remodeling / fibroblast growth factor receptor signaling pathway / cell fate commitment / canonical Wnt signaling pathway / chondrocyte differentiation / BMP signaling pathway / gastrulation / bone resorption / ossification / synaptic transmission, glutamatergic / axon guidance / wound healing / protein catabolic process / multicellular organism growth / cellular response to virus / regulation of blood pressure / vasodilation / gene expression / protein-containing complex assembly / protein heterodimerization activity Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | 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, 3 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_14582.map.gz | 79.2 MB | EMDB map data format | |
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Header (meta data) | emd-14582-v30.xml emd-14582.xml | 20.6 KB 20.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_14582_fsc.xml | 11.4 KB | Display | FSC data file |
Images | emd_14582.png | 182 KB | ||
Masks | emd_14582_msk_1.map | 125 MB | Mask map | |
Others | emd_14582_additional_1.map.gz emd_14582_half_map_1.map.gz emd_14582_half_map_2.map.gz | 98.7 MB 98.5 MB 98.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14582 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14582 | HTTPS FTP |
-Validation report
Summary document | emd_14582_validation.pdf.gz | 652.1 KB | Display | EMDB validaton report |
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Full document | emd_14582_full_validation.pdf.gz | 651.7 KB | Display | |
Data in XML | emd_14582_validation.xml.gz | 18.6 KB | Display | |
Data in CIF | emd_14582_validation.cif.gz | 24.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14582 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14582 | HTTPS FTP |
-Related structure data
Related structure data | 7zayMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_14582.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.84 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_14582_msk_1.map | ||||||||||||
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Density Histograms |
-Additional map: #1
File | emd_14582_additional_1.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_14582_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_14582_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Hetero-dimeric complex EXT1-EXT2
Entire | Name: Hetero-dimeric complex EXT1-EXT2 |
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Components |
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-Supramolecule #1: Hetero-dimeric complex EXT1-EXT2
Supramolecule | Name: Hetero-dimeric complex EXT1-EXT2 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 Details: local resolution-filtered map generated using a B-factor of -30 A2 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 160 KDa |
-Macromolecule #1: Exostosin-1
Macromolecule | Name: Exostosin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO EC number: glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 85.261859 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: GASRASRSHS RREEHSGRNG LHHPSPDHFW PRFPDALRPF VPWDQLENED SSVHISPRQK RDANSSIYKG KKCRMESCFD FTLCKKNGF KVYVYPQQKG EKIAESYQNI LAAIEGSRFY TSDPSQACLF VLSLDTLDRD QLSPQYVHNL RSKVQSLHLW N NGRNHLIF ...String: GASRASRSHS RREEHSGRNG LHHPSPDHFW PRFPDALRPF VPWDQLENED SSVHISPRQK RDANSSIYKG KKCRMESCFD FTLCKKNGF KVYVYPQQKG EKIAESYQNI LAAIEGSRFY TSDPSQACLF VLSLDTLDRD QLSPQYVHNL RSKVQSLHLW N NGRNHLIF NLYSGTWPDY TEDVGFDIGQ AMLAKASIST ENFRPNFDVS IPLFSKDHPR TGGERGFLKF NTIPPLRKYM LV FKGKRYL TGIGSDTRNA LYHVHNGEDV VLLTTCKHGK DWQKHKDSRC DRDNTEYEKY DYREMLHNAT FCLVPRGRRL GSF RFLEAL QAACVPVMLS NGWELPFSEV INWNQAAVIG DERLLLQIPS TIRSIHQDKI LALRQQTQFL WEAYFSSVEK IVLT TLEII QDRIFKHISR NSLIWNKHPG GLFVLPQYSS YLGDFPYYYA NLGLKPPSKF TAVIHAVTPL VSQSQPVLKL LVAAA KSQY CAQIIVLWNC DKPLPAKHRW PATAVPVVVI EGESKVMSSR FLPYDNIITD AVLSLDEDTV LSTTEVDFAF TVWQSF PER IVGYPARSHF WDNSKERWGY TSKWTNDYSM VLTGAAIYHK YYHYLYSHYL PASLKNMVDQ LANCEDILMN FLVSAVT KL PPIKVTQKKQ YKETMMGQTS RASRWADPDH FAQRQSCMNT FASWFGYMPL IHSQMRLDPV LFKDQVSILR KKYRDIER L NNNNNNGHHH HHHHH |
-Macromolecule #2: Exostosin-2
Macromolecule | Name: Exostosin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO EC number: glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 79.056836 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: GASPHSIESS NDWNVEKRSI RDVPVVRLPA DSPIPERGDL SCRMHTCFDV YRCGFNPKNK IKVYIYALKK YVDDFGVSVS NTISREYNE LLMAISDSDY YTDDINRACL FVPSIDVLNQ NTLRIKETAQ AMAQLSRWDR GTNHLLFNML PGGPPDYNTA L DVPRDRAL ...String: GASPHSIESS NDWNVEKRSI RDVPVVRLPA DSPIPERGDL SCRMHTCFDV YRCGFNPKNK IKVYIYALKK YVDDFGVSVS NTISREYNE LLMAISDSDY YTDDINRACL FVPSIDVLNQ NTLRIKETAQ AMAQLSRWDR GTNHLLFNML PGGPPDYNTA L DVPRDRAL LAGGGFSTWT YRQGYDVSIP VYSPLSAEVD LPEKGPGPRQ YFLLSSQVGL HPEYREDLEA LQVKHGESVL VL DKCTNLS EGVLSVRKRC HKHQVFDYPQ VLQEATFCVV LRGARLGQAV LSDVLQAGCV PVVIADSYIL PFSEVLDWKR ASV VVPEEK MSDVYSILQS IPQRQIEEMQ RQARWFWEAY FQSIKAIALA TLQIINDRIY PYAAISYEEW NDPPAVKWGS VSNP LFLPL IPPQSQGFTA IVLTYDRVES LFRVITEVSK VPSLSKLLVV WNNQNKNPPE DSLWPKIRVP LKVVRTAENK LSNRF FPYD EIETEAVLAI DDDIIMLTSD ELQFGYEVWR EFPDRLVGYP GRLHLWDHEM NKWKYESEWT NEVSMVLTGA AFYHKY FNY LYTYKMPGDI KNWVDAHMNC EDIAMNFLVA NVTGKAVIKV TPRKKFKCPE CTAIDGLSLD QTHMVERSEC INKFASV FG TMPLKVVEHR ADPVLYKDDF PEKLKSFPNI GSLNNNNNNG HHHHHHHH |
-Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Macromolecule #5: URIDINE-5'-DIPHOSPHATE
Macromolecule | Name: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: UDP |
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Molecular weight | Theoretical: 404.161 Da |
Chemical component information | ChemComp-UDP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.4 mg/mL |
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Buffer | pH: 7.2 |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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). |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 46.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |