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- PDB-9kqc: Chlorella virus Hyaluronan Synthase bound to VNAR -

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Basic information

Entry
Database: PDB / ID: 9kqc
TitleChlorella virus Hyaluronan Synthase bound to VNAR
Components
  • Hyaluronan synthase
  • VNAR
KeywordsTRANSFERASE / Chlorella virus Hyaluronan Synthase / membrane-integrated glycosyltransferase
Function / homologyhyaluronan synthase activity / Glycosyltransferase like family 2 / hyaluronan biosynthetic process / Nucleotide-diphospho-sugar transferases / plasma membrane / : / URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE / Hyaluronan synthase
Function and homology information
Biological speciesChlorella virus
Chiloscyllium plagiosum (whitespotted bambooshark)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsDeng, P. / Zhang, X. / Xu, M. / Wen, J. / Li, P. / Bi, Y. / Wang, H.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: J Struct Biol X / Year: 2025
Title: Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example.
Authors: Penghui Deng / Xiaoyue Zhang / Jianqing Wen / Mingce Xu / Pengwei Li / Hao Wang / Yunchen Bi /
Abstract: In cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is naturally devoid of light chains. The variable regions of IgNAR (VNARs) are solely responsible for antigen recognition, ...In cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is naturally devoid of light chains. The variable regions of IgNAR (VNARs) are solely responsible for antigen recognition, similar to VHHs (variable domain of the heavy chain of heavy-chain antibodies) in camelids. Although VNARs have attracted growing interest, generating VNARs against membrane proteins remains challenging. Furthermore, the structure of a VNAR in complex with a membrane protein has not yet been reported. This study features a membrane protein, Chlorella virus hyaluronan synthase (CvHAS), and provides a comprehensive methodological approach to generate its specific shark VNARs, addressing several major concerns and important optimizations. We showed that shark physiological urea pressure was tolerable for CvHAS, and indirect immobilization was strongly preferred over passive adsorption for membrane proteins. Together with optimizations to improve mononuclear cell (MC) viability and VNAR expression efficiency, we successfully generated S2F6, a CvHAS-specific VNAR with nM-level high affinity. The structure of the CvHAS-S2F6 complex was then determined by cryogenic electron microscopy (cryo-EM), reporting the first membrane protein and VNAR complex structure. It shows that S2F6 binds to the cytoplasmic domain of CvHAS, with a different epitope than the reported CvHAS-specific VHHs. This study provides valuable insights into developing VNARs for membrane proteins and their applications in structural biology.
History
DepositionNov 25, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 14, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 18, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Hyaluronan synthase
B: VNAR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,4734
Polymers79,8112
Non-polymers6622
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 Hyaluronan synthase


Mass: 65337.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlorella virus / Gene: CZ-2_118R, PBCVCZ2_118R / Production host: Escherichia coli (E. coli) / References: UniProt: M1H2Q1
#2: Protein VNAR


Mass: 14473.181 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chiloscyllium plagiosum (whitespotted bambooshark)
Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-UD1 / URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE


Mass: 607.354 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H27N3O17P2
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / 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: Chlorella virus Hyaluronan Synthase bound to VNAR. / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 79.708 kDa/nm / Experimental value: NO
Source (natural)Organism: Chlorella virus
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI MORGAGNI
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: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 263915 / Symmetry type: POINT

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