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- EMDB-52913: Human chondroitin sulfate polymerase complex CHSY3-CHPF -

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Basic information

Entry
Database: EMDB / ID: EMD-52913
TitleHuman chondroitin sulfate polymerase complex CHSY3-CHPF
Map dataCHSY3-CHPF composite map
Sample
  • Complex: Human chondroitin sulfate polymerase complex CHSY3-CHPF
    • Protein or peptide: Chondroitin sulfate synthase 2
    • Protein or peptide: Chondroitin sulfate synthase 3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: MANGANESE (II) ION
Keywordsglycosyltransferase / chondroitin sulfate / polymerization / heterodimeric complex / TRANSFERASE
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
Chondroitin sulfate synthase 3 / Chondroitin sulfate synthase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsDutta P / Cordeiro RL / Wild R
Funding support France, 4 items
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, 2025-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateDec 10, 2025-
Current statusDec 10, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52913.png

Downloads & links

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Map

FileDownload / File: emd_52913.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCHSY3-CHPF composite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 384 pix.
= 280.32 Å		0.73 Å/pix.
x 384 pix.
= 280.32 Å		0.73 Å/pix.
x 384 pix.
= 280.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.73 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0918
Minimum - Maximum-0.26812524 - 0.7824346
Average (Standard dev.)0.017294154 (±0.010566278)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 280.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human chondroitin sulfate polymerase complex CHSY3-CHPF

EntireName: Human chondroitin sulfate polymerase complex CHSY3-CHPF
Components
  • Complex: Human chondroitin sulfate polymerase complex CHSY3-CHPF
    • Protein or peptide: Chondroitin sulfate synthase 2
    • Protein or peptide: Chondroitin sulfate synthase 3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Human chondroitin sulfate polymerase complex CHSY3-CHPF

SupramoleculeName: Human chondroitin sulfate polymerase complex CHSY3-CHPF
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: In complex with UDP ligand and Mn2+ ions
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 165 KDa

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Macromolecule #1: Chondroitin sulfate synthase 2

MacromoleculeName: Chondroitin sulfate synthase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 78.8475 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPGGSTGGEN WEPRVLPYHP AQPGQAAKKA VRTRYISTEL GIRQRLLVAV LTSQTTLPTL GVAVNRTLGH RLERVVFLTG ARGRRAPPG MAVVTLGEER PIGHLHLALR HLLEQHGDDF DWFFLVPDTT YTEAHGLARL TGHLSLASAA HLYLGRPQDF I GGEPTPGR ...String:
GPGGSTGGEN WEPRVLPYHP AQPGQAAKKA VRTRYISTEL GIRQRLLVAV LTSQTTLPTL GVAVNRTLGH RLERVVFLTG ARGRRAPPG MAVVTLGEER PIGHLHLALR HLLEQHGDDF DWFFLVPDTT YTEAHGLARL TGHLSLASAA HLYLGRPQDF I GGEPTPGR YCHGGFGVLL SRMLLQQLRP HLEGCRNDIV SARPDEWLGR CILDATGVGC TGDHEGVHYS HLELSPGEPV QE GDPHFRS ALTAHPVRDP VHMYQLHKAF ARAELERTYQ EIQELQWEIQ NTSHLAVDGD QAAAWPVGIP APSRPASRFE VLR WDYFTE QHAFSCADGS PRCPLRGADR ADVADVLGTA LEELNRRYHP ALRLQKQQLV NGYRRFDPAR GMEYTLDLQL EALT PQGGR RPLTRRVQLL RPLSRVEILP VPYVTEASRL TVLLPLAAAE RDLAPGFLEA FATAALEPGD AAAALTLLLL YEPRQ AQRV AHADVFAPVK AHVAELERRF PGARVPWLSV QTAAPSPLRL MDLLSKKHPL DTLFLLAGPD TVLTPDFLNR CRMHAI SGW QAFFPMHFQA FHPAVAPPQG PGPPELGRDT GRFDRQAASE ACFYNSDYVA ARGRLAAASE QEEELLESLD VYELFLH FS SLHVLRAVEP ALLQRYRAQT CSARLSEDLY HRCLQSVLEG LGSRTQLAML LFEQEQGNST GTLEVLFQ

UniProtKB: Chondroitin sulfate synthase 2

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Macromolecule #2: Chondroitin sulfate synthase 3

MacromoleculeName: Chondroitin sulfate synthase 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 86.4565 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPGGSTGSGD GGAAAPSARP RDFLYVGVMT AQKYLGSRAL AAQRTWARFI PGRVEFFSSQ QPPNAGQPPP PLPVIALPGV DDSYPPQKK SFMMIKYMHD HYLDKYEWFM RADDDVYIKG DKLEEFLRSL NSSKPLYLGQ TGLGNIEELG KLGLEPGENF C MGGPGMIF ...String:
GPGGSTGSGD GGAAAPSARP RDFLYVGVMT AQKYLGSRAL AAQRTWARFI PGRVEFFSSQ QPPNAGQPPP PLPVIALPGV DDSYPPQKK SFMMIKYMHD HYLDKYEWFM RADDDVYIKG DKLEEFLRSL NSSKPLYLGQ TGLGNIEELG KLGLEPGENF C MGGPGMIF SREVLRRMVP HIGECLREMY TTHEDVEVGR CVRRFGGTQC VWSYEMQQLF HENYEHNRKG YIQDLHNSKI HA AITLHPN KRPAYQYRLH NYMLSRKISE LRYRTIQLHR ESALMSKLSN TEVSKEDQQL GVIPSFNHFQ PRERNEVIEW EFL TGKLLY SAAENQPPRQ SLSSILRTAL DDTVLQVMEM INENAKSRGR LIDFKEIQYG YRRVNPMHGV EYILDLLLLY KRHK GRKLT VPVRRHAYLQ QLFSKPFFRE TEELDVNSLV ESINSETQSF SFISNSLKIL SSFQGAKEMG GHNEKKVHIL VPLIG RYDI FLRFMENFEN MCLIPKQNVK LVIILFSRDS GQDSSKHIEL IKGYQNKYPK AEMTLIPMKG EFSRGLGLEM ASAQFD NDT LLLFCDVDLI FREDFLQRCR DNTIQGQQVY YPIIFSQYDP KVTNGGNPPT DDYFIFSKKT GFWRDYGYGI TCIYKSD LL GAGGFDTSIQ GWGLEDVDLY NKVILSGLRP FRSQEVGVVH IFHPVHCDPN LDPKQYKMCL GSKASTFAST MQLAELWL E KHLGVRYNRT LSGTGSGLND IFEAQKIEWH EG

UniProtKB: Chondroitin sulfate synthase 3

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #4: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

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

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Macromolecule #5: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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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

Concentration0.6 mg/mL
BufferpH: 6.5
Component:
ConcentrationFormulaName
20.0 mMC6H13NO4SMES
100.0 mMNaClsodium chloride
2.5 mMMnCl2Manganese chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 12169 / Average exposure time: 4.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4534470
CTF correctionSoftware - Name: cryoSPARC (ver. v3.3.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold 2 predicted model of CHSY3-CHPF complex
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.3.3.1) / Software - details: non-uniform refinement / Number images used: 166015
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.3.1) / Details: ab-initio reconstruction
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v.3.3.1)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: CHSY3-CHPF complex
RefinementProtocol: RIGID BODY FIT
Output model

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

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