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- EMDB-25036: Cryo-EM 3D map of the human Exostosin-1 and Exostosin-2 heterodim... -

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Entry
Database: EMDB / ID: EMD-25036
TitleCryo-EM 3D map of the human Exostosin-1 and Exostosin-2 heterodimer in complex with a 4-sugar oligosaccharide acceptor analog
Map data
Sample
  • Complex: hEXT1/2
    • Protein or peptide: Exostosin-1
    • Protein or peptide: Exostosin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: URIDINE-5'-DIPHOSPHATE
Keywordsexostosin1 / exostosin2 / glycosyltransferase / heparan sulfate / acceptor / TRANSFERASE
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 / chondrocyte hypertrophy / embryonic skeletal joint development / hematopoietic stem cell migration to bone marrow / fluid transport / tight junction organization / glucuronosyltransferase activity / limb joint morphogenesis / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / heparin proteoglycan biosynthetic process / sebaceous gland development / glomerular basement membrane development / response to heparin / stomach development / glycosaminoglycan biosynthetic process / HS-GAG biosynthesis / glandular epithelial cell differentiation / lymphocyte migration into lymphoid organs / sulfation / chondrocyte proliferation / dendritic cell migration / endochondral bone morphogenesis / dendrite self-avoidance / heparan sulfate proteoglycan biosynthetic process / acetylglucosaminyltransferase activity / endochondral bone growth / podocyte differentiation / hematopoietic stem cell homeostasis / cartilage development involved in endochondral bone morphogenesis / sodium ion homeostasis / basement membrane organization / vocalization behavior / endochondral ossification / vacuole organization / olfactory bulb development / cranial skeletal system development / multicellular organismal-level water homeostasis / leukocyte tethering or rolling / endoderm development / polysaccharide biosynthetic process / fear response / response to light intensity / optic nerve development / regulation of tumor necrosis factor-mediated signaling pathway / protein N-linked glycosylation / collagen fibril organization / neural crest cell differentiation / ossification involved in bone maturation / stem cell division / hair follicle morphogenesis / cell adhesion mediated by integrin / : / heart contraction / epithelial tube branching involved in lung morphogenesis / antigen processing and presentation / glycosyltransferase activity / mesoderm development / motor behavior / mesoderm formation / social behavior / cell fate commitment / blood vessel remodeling / catalytic complex / fibroblast growth factor receptor signaling pathway / hematopoietic stem cell differentiation / canonical Wnt signaling pathway / chondrocyte differentiation / BMP signaling pathway / bone resorption / gastrulation / cellular response to fibroblast growth factor stimulus / ossification / axon guidance / synaptic transmission, glutamatergic / wound healing / protein catabolic process / cellular response to virus / regulation of blood pressure / vasodilation / multicellular organism growth / protein-containing complex assembly / gene expression / protein heterodimerization activity / Golgi membrane / synapse
Similarity search - Function
Exostosin-like / Exostosin, GT47 domain / Exostosin GT47 domain / 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: 3.4 Å
AuthorsLi H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex.
Authors: Hua Li / Digantkumar Chapla / Robert A Amos / Annapoorani Ramiah / Kelley W Moremen / Huilin Li /
Abstract: Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-) co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix ...Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-) co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1-EXT2 (EXT1-2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold β1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. The two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.
History
DepositionSep 28, 2021-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25036.png

Downloads & links

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Map

FileDownload / File: emd_25036.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0109
Minimum - Maximum-0.0017307906 - 2.1653304
Average (Standard dev.)0.002322152 (±0.035610627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25036_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_25036_additional_1.map
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Half map: #1

Fileemd_25036_half_map_1.map
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Half map: #2

Fileemd_25036_half_map_2.map
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Sample components

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Entire : hEXT1/2

EntireName: hEXT1/2
Components
  • Complex: hEXT1/2
    • 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: hEXT1/2

SupramoleculeName: hEXT1/2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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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: 83.404023 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: GFRASRSHSR REEHSGRNGL HHPSPDHFWP RFPDALRPFV PWDQLENEDS SVHISPRQKR DANSSIYKGK KCRMESCFDF TLCKKNGFK VYVYPQQKGE KIAESYQNIL AAIEGSRFYT SDPSQACLFV LSLDTLDRDQ LSPQYVHNLR SKVQSLHLWN N GRNHLIFN ...String:
GFRASRSHSR REEHSGRNGL HHPSPDHFWP RFPDALRPFV PWDQLENEDS SVHISPRQKR DANSSIYKGK KCRMESCFDF TLCKKNGFK VYVYPQQKGE KIAESYQNIL AAIEGSRFYT SDPSQACLFV LSLDTLDRDQ LSPQYVHNLR SKVQSLHLWN N GRNHLIFN LYSGTWPDYT EDVGFDIGQA MLAKASISTE NFRPNFDVSI PLFSKDHPRT GGERGFLKFN TIPPLRKYML VF KGKRYLT GIGSDTRNAL YHVHNGEDVV LLTTCKHGKD WQKHKDSRCD RDNTEYEKYD YREMLHNATF CLVPRGRRLG SFR FLEALQ AACVPVMLSN GWELPFSEVI NWNQAAVIGD ERLLLQIPST IRSIHQDKIL ALRQQTQFLW EAYFSSVEKI VLTT LEIIQ DRIFKHISRN SLIWNKHPGG LFVLPQYSSY LGDFPYYYAN LGLKPPSKFT AVIHAVTPLV SQSQPVLKLL VAAAK SQYC AQIIVLWNCD KPLPAKHRWP ATAVPVVVIE GESKVMSSRF LPYDNIITDA VLSLDEDTVL STTEVDFAFT VWQSFP ERI VGYPARSHFW DNSKERWGYT SKWTNDYSMV LTGAAIYHKY YHYLYSHYLP ASLKNMVDQL ANCEDILMNF LVSAVTK LP PIKVTQKKQY KETMMGQTSR ASRWADPDHF AQRQSCMNTF ASWFGYMPLI HSQMRLDPVL FKDQVSILRK KYRDIERL

UniProtKB: Exostosin-1

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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: 77.180984 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: WPHSIESSND WNVEKRSIRD VPVVRLPADS PIPERGDLSC RMHTCFDVYR CGFNPKNKIK VYIYALKKYV DDFGVSVSNT ISREYNELL MAISDSDYYT DDINRACLFV PSIDVLNQNT LRIKETAQAM AQLSRWDRGT NHLLFNMLPG GPPDYNTALD V PRDRALLA ...String:
WPHSIESSND WNVEKRSIRD VPVVRLPADS PIPERGDLSC RMHTCFDVYR CGFNPKNKIK VYIYALKKYV DDFGVSVSNT ISREYNELL MAISDSDYYT DDINRACLFV PSIDVLNQNT LRIKETAQAM AQLSRWDRGT NHLLFNMLPG GPPDYNTALD V PRDRALLA GGGFSTWTYR QGYDVSIPVY SPLSAEVDLP EKGPGPRQYF LLSSQVGLHP EYREDLEALQ VKHGESVLVL DK CTNLSEG VLSVRKRCHK HQVFDYPQVL QEATFCVVLR GARLGQAVLS DVLQAGCVPV VIADSYILPF SEVLDWKRAS VVV PEEKMS DVYSILQSIP QRQIEEMQRQ ARWFWEAYFQ SIKAIALATL QIINDRIYPY AAISYEEWND PPAVKWGSVS NPLF LPLIP PQSQGFTAIV LTYDRVESLF RVITEVSKVP SLSKLLVVWN NQNKNPPEDS LWPKIRVPLK VVRTAENKLS NRFFP YDEI ETEAVLAIDD DIIMLTSDEL QFGYEVWREF PDRLVGYPGR LHLWDHEMNK WKYESEWTNE VSMVLTGAAF YHKYFN YLY TYKMPGDIKN WVDAHMNCED IAMNFLVANV TGKAVIKVTP RKKFKCPECT AIDGLSLDQT HMVERSECIN KFASVFG TM PLKVVEHRAD PVLYKDDFPE KLKSFPNIGS L

UniProtKB: Exostosin-2

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #6: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

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

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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

Concentration0.3 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPESHEPES
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 299 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 193.0 K / Max: 193.0 K
Alignment procedureComa free - Residual tilt: 0.05 mrad
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3080 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1938290
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 91157
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7scj:
Cryo-EM structure of the human Exostosin-1 and Exostosin-2 heterodimer in complex with a 4-sugar oligosaccharide acceptor analog

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