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- PDB-7au2: Cryo-EM structure of human exostosin-like 3 (EXTL3) -

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Basic information

Entry
Database: PDB / ID: 7au2
TitleCryo-EM structure of human exostosin-like 3 (EXTL3)
ComponentsExostosin-like 3
KeywordsTRANSFERASE / glycosyltransferase / heparan / n-acetylglucosaminyltransferase
Function / homology
Function and homology information


glucuronosyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / positive regulation of detection of glucose / protein-hormone receptor activity / positive regulation of keratinocyte proliferation / negative regulation of keratinocyte differentiation / heparan sulfate proteoglycan biosynthetic process / negative regulation of inflammatory response to wounding / XBP1(S) activates chaperone genes / : ...glucuronosyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / positive regulation of detection of glucose / protein-hormone receptor activity / positive regulation of keratinocyte proliferation / negative regulation of keratinocyte differentiation / heparan sulfate proteoglycan biosynthetic process / negative regulation of inflammatory response to wounding / XBP1(S) activates chaperone genes / : / glycosyltransferase activity / negative regulation of cytokine production involved in inflammatory response / negative regulation of inflammatory response / positive regulation of cell growth / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / endoplasmic reticulum membrane / magnesium ion binding / endoplasmic reticulum / Golgi apparatus / nucleus / plasma membrane
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
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsWilson, L.F.L. / Dendooven, T. / Hardwick, S.W. / Chirgadze, D.Y. / Luisi, B.F. / Logan, D.T. / Mani, K. / Dupree, P.
Funding support United Kingdom, Sweden, 8items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
Wellcome Trust200873/Z/16/Z United Kingdom
Swedish Research Council2014-03402 Sweden
Swedish Research Council2016-04855 Sweden
Cancerfonden180710 Sweden
Engineering and Physical Sciences Research Council United Kingdom
University of Cambridge United Kingdom
OpenPlantBB/L014130/1 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis.
Authors: L F L Wilson / T Dendooven / S W Hardwick / A Echevarría-Poza / T Tryfona / K B R M Krogh / D Y Chirgadze / B F Luisi / D T Logan / K Mani / P Dupree /
Abstract: Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of ...Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating β-D-glucuronosyl and N-acetyl-α-D-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)-each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3's GT47 domain to transfer β-D-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.
History
DepositionNov 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 18, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 13, 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-like 3
B: Exostosin-like 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,7566
Polymers203,4102
Non-polymers2,3464
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12920 Å2
ΔGint-21 kcal/mol
Surface area52010 Å2
MethodPISA

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Components

#1: Protein Exostosin-like 3 / EXT-related protein 1 / Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase ...EXT-related protein 1 / Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / Hereditary multiple exostoses gene isolog / Multiple exostosis-like protein 3 / Putative tumor suppressor protein EXTL3


Mass: 101705.117 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXTL3, EXTL1L, EXTR1, KIAA0519 / Cell line (production host): HEK-293 EBNA / Production host: Homo sapiens (human)
References: UniProt: O43909, glucuronosyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
Has ligand of interestN
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: Homodimer of EXTL3 globular domain / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 71.4 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
4Gctf1.06CTF correction
7Buccaneermodel fitting
8Cootmodel fitting
9ISOLDEmodel fitting
11PHENIX1.18.2model refinement
12RELION2.1initial Euler assignment
13RELION2.1final Euler assignment
14RELION2.1classification
15RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 656292
3D reconstructionResolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171285 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511556
ELECTRON MICROSCOPYf_angle_d0.65515730
ELECTRON MICROSCOPYf_dihedral_angle_d21.7881600
ELECTRON MICROSCOPYf_chiral_restr0.0471752
ELECTRON MICROSCOPYf_plane_restr0.0052004

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