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- PDB-9yq2: Chlorella virus hyaluronan synthase bound to DDM -

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Basic information

Entry
Database: PDB / ID: 9yq2
TitleChlorella virus hyaluronan synthase bound to DDM
Components
  • Hyaluronan synthase
  • nanobody Nb872
  • nanobody Nb881
KeywordsTRANSFERASE/IMMUNE SYSTEM / Membrane protein / glycosyltransferase / detergent / TRANSFERASE-IMMUNE SYSTEM complex
Function / homologyhyaluronan synthase activity / Glycosyltransferase like family 2 / hyaluronan biosynthetic process / Nucleotide-diphospho-sugar transferases / plasma membrane / : / CHOLESTEROL HEMISUCCINATE / Hyaluronan synthase
Function and homology information
Biological speciesParamecium bursaria Chlorella virus CZ-2
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsStephens, Z. / Zimmer, J.
Funding support United States, 1items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hyaluronan synthase
B: nanobody Nb872
C: nanobody Nb881
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,7467
Polymers95,6393
Non-polymers1,1074
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 2 molecules BC

#2: Antibody nanobody Nb872


Mass: 14783.248 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Antibody nanobody Nb881


Mass: 14966.389 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria)

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Hyaluronan synthase


Mass: 65889.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paramecium bursaria Chlorella virus CZ-2
Gene: CZ-2_118R / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: M1H2Q1, hyaluronan synthase
#6: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H46O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM

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Non-polymers , 2 types, 3 molecules

#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn

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Details

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: Ternary complex of CvHAS bound to Nb872 and Nb881 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Paramecium bursaria Chlorella virus CZ-2
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 513368 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026088
ELECTRON MICROSCOPYf_angle_d0.4448267
ELECTRON MICROSCOPYf_dihedral_angle_d5.341842
ELECTRON MICROSCOPYf_chiral_restr0.04907
ELECTRON MICROSCOPYf_plane_restr0.0031020

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