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- EMDB-14582: Human heparan sulfate polymerase complex EXT1-EXT2 -

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Basic information

Entry
Database: EMDB / ID: EMD-14582
TitleHuman heparan sulfate polymerase complex EXT1-EXT2
Map data
Sample
  • Complex: Hetero-dimeric complex EXT1-EXT2
    • Protein or peptide: Exostosin-1
    • Protein or peptide: Exostosin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: URIDINE-5'-DIPHOSPHATE
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 / chondroitin sulfate metabolic process / UDP-N-acetylglucosamine transferase complex / 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 / dendrite self-avoidance / heparan sulfate proteoglycan biosynthetic process / HS-GAG biosynthesis / glycosaminoglycan biosynthetic process / lymphocyte migration into lymphoid organs / chondrocyte proliferation / sulfation / hematopoietic stem cell homeostasis / podocyte differentiation / glandular epithelial cell differentiation / dendritic cell migration / endochondral bone morphogenesis / polysaccharide biosynthetic process / endochondral bone growth / sodium ion homeostasis / acetylglucosaminyltransferase activity / response to light intensity / basement membrane organization / cartilage development involved in endochondral bone morphogenesis / cranial skeletal system development / vocalization behavior / stem cell division / endoderm development / multicellular organismal-level water homeostasis / leukocyte tethering or rolling / vacuole organization / olfactory bulb development / endochondral ossification / fear response / optic nerve development / protein N-linked glycosylation / cellular response to fibroblast growth factor stimulus / neural crest cell differentiation / collagen fibril organization / ossification involved in bone maturation / motor behavior / cell adhesion mediated by integrin / epithelial tube branching involved in lung morphogenesis / hair follicle morphogenesis / heart contraction / glycosyltransferase activity / social behavior / protein glycosylation / mesoderm development / mesoderm formation / catalytic complex / antigen processing and presentation / gastrulation / canonical Wnt signaling pathway / hematopoietic stem cell differentiation / blood vessel remodeling / fibroblast growth factor receptor signaling pathway / cell fate commitment / chondrocyte differentiation / BMP signaling pathway / bone resorption / ossification / synaptic transmission, glutamatergic / axon guidance / protein catabolic process / multicellular organism growth / wound healing / cellular response to virus / regulation of blood pressure / vasodilation / gene expression / protein-containing complex assembly / protein heterodimerization activity
Similarity search - Function
Exostosin-like / Exostosin, GT47 domain / Exostosin family / Glycosyl transferase 64 domain / Glycosyl transferase family 64 domain / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Exostosin-1 / Exostosin-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsLeisico F / Omeiri J / Hons M / Schoehn G / Lortat-Jacob H / Wild R
Funding support France, 3 items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS)ATIP-Avenir France
Agence Nationale de la Recherche (ANR)ANR-18-CE11- 0006-01 France
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-02 France
CitationJournal: 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
DepositionMar 23, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateDec 7, 2022-
Current statusDec 7, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14582.png

Downloads & links

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Map

FileDownload / File: emd_14582.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 320 pix.
= 268.8 Å		0.84 Å/pix.
x 320 pix.
= 268.8 Å		0.84 Å/pix.
x 320 pix.
= 268.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.09967563 - 0.13719282
Average (Standard dev.)3.3763044e-06 (±0.0023645854)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14582_msk_1.map
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Additional map: #1

Fileemd_14582_additional_1.map
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Half map: #2

Fileemd_14582_half_map_1.map
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Half map: #1

Fileemd_14582_half_map_2.map
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Sample components

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Entire : Hetero-dimeric complex EXT1-EXT2

EntireName: Hetero-dimeric complex EXT1-EXT2
Components
  • Complex: Hetero-dimeric complex EXT1-EXT2
    • Protein or peptide: Exostosin-1
    • Protein or peptide: Exostosin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: URIDINE-5'-DIPHOSPHATE

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Supramolecule #1: Hetero-dimeric complex EXT1-EXT2

SupramoleculeName: 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
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 160 KDa

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Macromolecule #1: Exostosin-1

MacromoleculeName: Exostosin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85.261859 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: 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

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Macromolecule #2: Exostosin-2

MacromoleculeName: Exostosin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 79.056836 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: 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

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

ChemComp-UDP:
URIDINE-5'-DIPHOSPHATE / UDP*YM / Uridine diphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsPurified 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).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 46.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Model of the hetero-dimeric EXT1-EXT2 complex predicted using the software AlphaFold2.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 286390
FSC plot (resolution estimation)

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