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- PDB-7sch: Cryo-EM structure of the human Exostosin-1 and Exostosin-2 heterodimer -

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Basic information

Entry
Database: PDB / ID: 7sch
TitleCryo-EM structure of the human Exostosin-1 and Exostosin-2 heterodimer
Components
  • Exostosin-1
  • Exostosin-2
KeywordsTRANSFERASE / exostosin1 / exostosin2 / glycosyltransferase / heparan sulfate
Function / homology
Function and homology information


glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / UDP-N-acetylglucosamine transferase complex / glucuronosyltransferase activity / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / glycosaminoglycan biosynthetic process ...glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / UDP-N-acetylglucosamine transferase complex / glucuronosyltransferase activity / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / glycosaminoglycan biosynthetic process / HS-GAG biosynthesis / heparan sulfate proteoglycan biosynthetic process / acetylglucosaminyltransferase activity / polysaccharide biosynthetic process / glycosyltransferase activity / catalytic complex / ossification / protein heterodimerization activity / Golgi membrane / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / protein homodimerization activity / extracellular exosome / membrane / metal ion binding
Similarity search - Function
Exostosin-like / Exostosin, GT47 domain / Exostosin GT47 domain / Glycosyl transferase 64 domain / Glycosyl transferase family 64 domain / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Nucleotide-diphospho-sugar transferases / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Exostosin-1 / Exostosin-2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLi, H. / Li, H.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2May 17, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Exostosin-1
B: Exostosin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,2293
Polymers160,6422
Non-polymers5871
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Exostosin-1 / Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N- ...Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / Multiple exostoses protein 1 / Putative tumor suppressor protein EXT1


Mass: 83404.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXT1 / Production host: Mammalia (mammals)
References: UniProt: Q16394, glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase, N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase
#2: Protein Exostosin-2 / Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N- ...Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / Multiple exostoses protein 2 / Putative tumor suppressor protein EXT2


Mass: 77238.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXT2 / Production host: Mammalia (mammals)
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


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
Has ligand of interestY
Has protein modificationY

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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: hEXT1/2 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalia (mammals)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPESHEPES1
SpecimenConc.: 0.3 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 R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 299 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 193 K / Temperature (min): 193 K / Residual tilt: 0.05 mradians
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 66.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2980
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5186047
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120216 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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