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- PDB-9q8z: Human chondroitin sulfate polymerase complex CHSY3-CHPF -

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Basic information

Entry
Database: PDB / ID: 9q8z
TitleHuman chondroitin sulfate polymerase complex CHSY3-CHPF
Components
  • Chondroitin sulfate synthase 2
  • Chondroitin sulfate synthase 3
KeywordsTRANSFERASE / glycosyltransferase / chondroitin sulfate / polymerization / heterodimeric complex
Function / homology
Function and homology information


glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase / glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase activity / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase activity / CS-GAG biosynthesis / chondroitin sulfate proteoglycan biosynthetic process / Golgi cisterna membrane / mitochondrial matrix / Golgi membrane / metal ion binding / cytosol
Similarity search - Function
Chondroitin N-acetylgalactosaminyltransferase / : / Chondroitin N-acetylgalactosaminyltransferase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
: / URIDINE-5'-DIPHOSPHATE / Chondroitin sulfate synthase 3 / Chondroitin sulfate synthase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsDutta, P. / Cordeiro, R.L. / Wild, R.
Funding support France, 4items
OrganizationGrant numberCountry
Other privateImpulscience Program of the Bettencourt Schueller Foundation
Agence Nationale de la Recherche (ANR)ANR-23-CE44-0011 France
Laboratories of Excellence (LabEx)ANR-17-EURE-0003 France
Agence Nationale de la Recherche (ANR)ANR-21-ESRE-0046 France
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for human chondroitin sulfate chain polymerization.
Authors: Poushalee Dutta / Rosa L Cordeiro / Mélanie Friedel-Arboleas / Marie Bourgeais / Sylvain D Vallet / Margot Weber / Margaux Molinas / Huazhang Shu / Magnus N N Grønset / Rebecca L Miller / ...Authors: Poushalee Dutta / Rosa L Cordeiro / Mélanie Friedel-Arboleas / Marie Bourgeais / Sylvain D Vallet / Margot Weber / Margaux Molinas / Huazhang Shu / Magnus N N Grønset / Rebecca L Miller / Elisabetta Boeri Erba / Rebekka Wild /
Abstract: Chondroitin sulfates are complex polysaccharide chains that regulate various biological processes at the cell surface and within the extracellular matrix. Here, we identify four heterodimeric ...Chondroitin sulfates are complex polysaccharide chains that regulate various biological processes at the cell surface and within the extracellular matrix. Here, we identify four heterodimeric complexes responsible for chondroitin sulfate chain polymerization in humans: CHSY1-CHPF, CHSY1-CHPF2, CHSY3-CHPF, and CHSY3-CHPF2. Using a custom-tailored in vitro glycosylation assay based on chemo-enzymatically synthesized fluorescent substrates, we demonstrate that all four complexes exhibit chain polymerization activity. The cryo-EM structure of the CHSY3-CHPF complex provides molecular insights into the chondroitin sulfate chain polymerization reaction. The architecture of the catalytic sites suggests that CHSY1 and CHSY3 are enzymatically active, while CHPF and CHPF2 primarily play a stabilizing role. Mutational analysis of purified enzyme complexes, combined with an in cellulo complementation assay, confirms that only CHSY1 and CHSY3 have bifunctional glycosyltransferase activities. Based on the spatial arrangement of the catalytic sites, we propose that chondroitin sulfate chain polymerization follows a non-processive, distributive mechanism.
History
DepositionFeb 25, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 3, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 3, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 3, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 3, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Dec 3, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 3, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Dec 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Jan 7, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jan 7, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chondroitin sulfate synthase 2
B: Chondroitin sulfate synthase 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,4828
Polymers165,3042
Non-polymers1,1786
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Chondroitin sulfate synthase 2 / Chondroitin glucuronyltransferase 2 / Chondroitin-polymerizing factor / ChPF / Glucuronosyl-N- ...Chondroitin glucuronyltransferase 2 / Chondroitin-polymerizing factor / ChPF / Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase II / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase II / N-acetylgalactosaminyltransferase 2


Mass: 78847.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHPF, CSS2, UNQ651/PRO1281 / Cell line (production host): Freestyle 293-F / Production host: Homo sapiens (human)
References: UniProt: Q8IZ52, glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase, N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase
#2: Protein Chondroitin sulfate synthase 3 / Carbohydrate synthase 2 / Chondroitin glucuronyltransferase 3 / Chondroitin synthase 2 / ChSy-2 / ...Carbohydrate synthase 2 / Chondroitin glucuronyltransferase 3 / Chondroitin synthase 2 / ChSy-2 / Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase II / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase 3 / N-acetylgalactosaminyltransferase 3


Mass: 86456.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHSY3, CHSY2, CSS3 / Cell line (production host): FreeStyle 293-F / Production host: Homo sapiens (human)
References: UniProt: Q70JA7, glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase, N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#5: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
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: Human chondroitin sulfate polymerase complex CHSY3-CHPF
Type: COMPLEX / Details: In complex with UDP ligand and Mn2+ ions / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.165 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: FreeStyle 293-F
Buffer solutionpH: 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMESC6H13NO4S1
2100 mMsodium chlorideNaCl1
32.5 mMManganese chlorideMnCl21
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1600 nm
Image recordingAverage exposure time: 4.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12169

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.1particle selection
2EPUimage acquisition
4cryoSPARCv3.3.1CTF correction
7Cootmodel fitting
9cryoSPARCv3.3.1initial Euler assignment
10cryoSPARCv.3.3.1final Euler assignment
12cryoSPARCv.3.3.13D reconstructionnon-uniform refinement
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4534470
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166015 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingDetails: CHSY3-CHPF complex / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.02 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00299848
ELECTRON MICROSCOPYf_angle_d0.543113341
ELECTRON MICROSCOPYf_chiral_restr0.03991447
ELECTRON MICROSCOPYf_plane_restr0.00471727
ELECTRON MICROSCOPYf_dihedral_angle_d3.72271334

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