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- EMDB-73324: Chlorella virus hyaluronan synthase bound to an inserted UDP-GlcA -

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Basic information

Entry
Database: EMDB / ID: EMD-73324
TitleChlorella virus hyaluronan synthase bound to an inserted UDP-GlcA
Map data
Sample
  • Complex: Ternary complex of CvHAS, Nb872 and Nb881
    • Protein or peptide: Hyaluronan synthase
    • Protein or peptide: nanobody Nb872
    • Protein or peptide: nanobody Nb881
  • Ligand: URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: MANGANESE (II) ION
KeywordsGlycosyltransferase / Membrane Protein / Hyaluronan Synthase / Substrate / TRANSFERASE-IMMUNE SYSTEM complex
Function / homologyhyaluronan synthase activity / Glycosyltransferase like family 2 / hyaluronan biosynthetic process / Nucleotide-diphospho-sugar transferases / plasma membrane / Hyaluronan synthase
Function and homology information
Biological speciesParamecium bursaria Chlorella virus CZ-2 / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsStephens Z / Zimmer J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35 GM144130 United States
CitationJournal: bioRxiv / Year: 2025
Title: Insights into substrate binding and utilization by hyaluronan synthase.
Authors: Zachery Stephens / Julia Karasinska / Jochen Zimmer /
Abstract: Hyaluronan (HA) is an essential polysaccharide of the vertebrate extracellular matrix. It serves as an adhesive, lubricant, signaling molecule, and spatial filler without which embryogenesis would ...Hyaluronan (HA) is an essential polysaccharide of the vertebrate extracellular matrix. It serves as an adhesive, lubricant, signaling molecule, and spatial filler without which embryogenesis would not complete. HA is synthesized by a membrane-integrated glycosyltransferase (HAS) that polymerizes UDP-activated N-acetylglucosamine and glucuronic acid (GlcA) in an alternating fashion. The nascent HA chain is secreted across the plasma membrane during this process. How HAS couples these tasks remains poorly understood. Here, we employ a combination of structural biology, biochemistry and glycobiology to delineate how HAS recognizes and utilizes UDP-GlcA. Using single-particle cryo-EM, we reveal a two-step process by which HAS binds its substrate UDP-GlcA. Prior to proper insertion into the catalytic pocket, the substrate is bound in a proofreading pose that may increase substrate selectivity. This state is accompanied by conformational changes of active site residues surrounding the UDP-binding pocket and involves a pair of basic residues that sense the substrate's carboxyl group. Further, we establish that HAS is unable to catalyze UDP-GlcA turnover in the absence of an acceptor sugar, emphasizing the role of a priming GlcNAc in glycosyl transfer. Lastly, cryo-EM snapshots of a dodecylmaltoside molecule trapped in the active site provide novel insights into substrate promiscuity. Here, our studies demonstrate that HAS catalyzes semi-selective GlcA-transfer to non-canonical β-linked acceptors.
History
DepositionOct 15, 2025-
Header (metadata) releaseDec 10, 2025-
Map releaseDec 10, 2025-
UpdateDec 10, 2025-
Current statusDec 10, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73324.png

Downloads & links

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Map

FileDownload / File: emd_73324.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.673
Minimum - Maximum-3.3316607 - 4.3060737
Average (Standard dev.)-0.00040909825 (±0.093906455)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_73324_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_73324_half_map_2.map
Projections & Slices
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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of CvHAS, Nb872 and Nb881

EntireName: Ternary complex of CvHAS, Nb872 and Nb881
Components
  • Complex: Ternary complex of CvHAS, Nb872 and Nb881
    • Protein or peptide: Hyaluronan synthase
    • Protein or peptide: nanobody Nb872
    • Protein or peptide: nanobody Nb881
  • Ligand: URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Ternary complex of CvHAS, Nb872 and Nb881

SupramoleculeName: Ternary complex of CvHAS, Nb872 and Nb881 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Paramecium bursaria Chlorella virus CZ-2

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Macromolecule #1: Hyaluronan synthase

MacromoleculeName: Hyaluronan synthase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: hyaluronan synthase
Source (natural)Organism: Paramecium bursaria Chlorella virus CZ-2
Molecular weightTheoretical: 65.889078 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGTSWRTIVS ANLFAVGGAL LMLAPAIVGY VFQWNIGVSA VWGISVYGVF VLGFYIAQIV FSEFNRMRLS DWISLRPDNW NATRVAVII AGYREDPFMF KKCLESVRDS EYGNVARLIC VIDGDEEEDL KMAEIYKQVY NDNVKKPGVV LCESENKNGS T IDSDVSKN ...String:
MGTSWRTIVS ANLFAVGGAL LMLAPAIVGY VFQWNIGVSA VWGISVYGVF VLGFYIAQIV FSEFNRMRLS DWISLRPDNW NATRVAVII AGYREDPFMF KKCLESVRDS EYGNVARLIC VIDGDEEEDL KMAEIYKQVY NDNVKKPGVV LCESENKNGS T IDSDVSKN ICILQPHRGK RESLYTGFQL ASMDPSVHAV VLIDSDTVLE KNAILEVVYP LSCDPNIKAV AGECKIWNTD TI LSMLVSW RYFSAFNVER GAQSLWKTVQ CVGGPLGAYT IDIINEIKDP WITQTFLGNK CTYGDNRRLT NEVLMRGKKI VYT PFAVGW SDSPTNVMRY IVQQTRWSKS WCREIWYTLG SAWKHGFSGI YLAFECMYQI MYFFLVMYLF SYIAIKADIR AQTA TVLVS TLVTIIKSSY LALRAKNLKA FYFVLYTYVY FFCMIPARIT AMFTMFDIAW GTRGGNAKMT IGARVWLWAK QFLIT YMWW AGVLAAGVYS IVDNWYFDWA DIQYRFALVG ICSYLVFVSI VLVIYLIGKI TTWNYTPLQK ELIEERYLHN ASENAP EVL EHHHHHHHHH H

UniProtKB: Hyaluronan synthase

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Macromolecule #2: nanobody Nb872

MacromoleculeName: nanobody Nb872 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.783248 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLKV SCAASGRAFK TYRMAWFRQA PGKEREFVSG ISALETTYYA DSVKGRFTIS RDNTKNTVSL QMDSLKPED TAVYYCAARR YGGTDYTTTG SYDYWGQGTQ VTVSSHHHHH HEPEA

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Macromolecule #3: nanobody Nb881

MacromoleculeName: nanobody Nb881 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.956369 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLRL ACAASGRIFS SDTLAWFRRA PGKEREFVAA SRWSGGGTDY ADSVKGRFTF SRDNTFNTMC LEMNSLKPE DTAVYYCALR TARDSYYYTR NPTGYDYWGQ GTQSSHHHHH HEPEA

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Macromolecule #4: URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID

MacromoleculeName: URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID / type: ligand / ID: 4 / Number of copies: 1 / Formula: UGA
Molecular weightTheoretical: 580.285 Da
Chemical component information

ChemComp-UGA:
URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 110407
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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