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- PDB-8sne: Chlorella virus Hyaluronan Synthase bound to GlcA extended GlcNAc... -

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Basic information

Entry
Database: PDB / ID: 8sne
TitleChlorella virus Hyaluronan Synthase bound to GlcA extended GlcNAc primer and UDP
Components
  • Hyaluronan synthase
  • Nanobody 872
  • Nanobody 886
KeywordsTRANSFERASE / hyaluronic acid / hyaluronan / HA / HAS / glycosyltransferase / GT / membrane protein / nanobody / n-acetylglucosamine / glucuronic acid
Function / homology
Function and homology information


hyaluronan synthase activity / hyaluronan biosynthetic process / plasma membrane
Similarity search - Function
Glycosyltransferase like family 2 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
1,2-Distearoyl-sn-glycerophosphoethanolamine / : / URIDINE-5'-DIPHOSPHATE / CHOLESTEROL HEMISUCCINATE / Hyaluronan synthase
Similarity search - Component
Biological speciesParamecium bursaria Chlorella virus CZ-2
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsStephens, Z. / Zimmer, J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144130 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into translocation and tailored synthesis of hyaluronan.
Authors: Ireneusz Górniak / Zachery Stephens / Satchal K Erramilli / Tomasz Gawda / Anthony A Kossiakoff / Jochen Zimmer /
Abstract: Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons ...Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.
History
DepositionApr 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Nov 27, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.3Feb 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hyaluronan synthase
B: Nanobody 872
C: Nanobody 886
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,5579
Polymers95,4113
Non-polymers2,1466
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Antibody , 2 types, 2 molecules BC

#2: Antibody Nanobody 872


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 K-12 (bacteria)
#3: Antibody Nanobody 886


Mass: 14737.267 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli K-12 (bacteria)

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Hyaluronan synthase


Mass: 65890.062 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
#4: Polysaccharide beta-D-glucopyranuronic acid-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 397.332 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpAb1-3DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O][a2122A-1b_1-5]/1-2/a3-b1WURCSPDB2Glycan 1.1.0
[][b-D-GlcpNAc]{[(3+1)][b-D-GlcpA]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 5 molecules

#5: 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
#6: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#7: Chemical ChemComp-3PE / 1,2-Distearoyl-sn-glycerophosphoethanolamine / 3-SN-PHOSPHATIDYLETHANOLAMINE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE


Mass: 748.065 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H82NO8P / Comment: phospholipid*YM
#8: 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 Chlorella virus hyaluronan synthase bound to nanobodies 872 and 886
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 95.3 kDa/nm / Experimental value: NO
Source (natural)Organism: Paramecium bursaria Chlorella virus 1
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220556 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046169
ELECTRON MICROSCOPYf_angle_d0.5248373
ELECTRON MICROSCOPYf_dihedral_angle_d6.767870
ELECTRON MICROSCOPYf_chiral_restr0.042922
ELECTRON MICROSCOPYf_plane_restr0.0041028

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