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- EMDB-62500: Chlorella virus Hyaluronan Synthase bound to VNAR -

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Basic information

Entry
Database: EMDB / ID: EMD-62500
TitleChlorella virus Hyaluronan Synthase bound to VNAR
Map data
Sample
  • Complex: Chlorella virus Hyaluronan Synthase bound to VNAR.
    • Protein or peptide: Hyaluronan synthase
    • Protein or peptide: VNAR
  • Ligand: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE
  • Ligand: MANGANESE (II) ION
KeywordsChlorella virus Hyaluronan Synthase / membrane-integrated glycosyltransferase / TRANSFERASE
Function / homologyhyaluronan synthase activity / Glycosyltransferase like family 2 / hyaluronan biosynthetic process / Nucleotide-diphospho-sugar transferases / plasma membrane / Hyaluronan synthase
Function and homology information
Biological speciesChlorella virus / Chiloscyllium plagiosum (whitespotted bambooshark)
Methodsingle 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, 1 items
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, 2024-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62500.png

Downloads & links

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Map

FileDownload / File: emd_62500.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.46 Å/pix.
x 420 pix.
= 193.2 Å		0.46 Å/pix.
x 420 pix.
= 193.2 Å		0.46 Å/pix.
x 420 pix.
= 193.2 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.46 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.043907374 - 0.0895769
Average (Standard dev.)0.00068829104 (±0.003580454)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 193.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_62500_half_map_1.map
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Half map: #1

Fileemd_62500_half_map_2.map
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Sample components

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Entire : Chlorella virus Hyaluronan Synthase bound to VNAR.

EntireName: Chlorella virus Hyaluronan Synthase bound to VNAR.
Components
  • Complex: Chlorella virus Hyaluronan Synthase bound to VNAR.
    • Protein or peptide: Hyaluronan synthase
    • Protein or peptide: VNAR
  • Ligand: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Chlorella virus Hyaluronan Synthase bound to VNAR.

SupramoleculeName: Chlorella virus Hyaluronan Synthase bound to VNAR. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Chlorella virus
Molecular weightTheoretical: 79.708 kDa/nm

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Macromolecule #1: Hyaluronan synthase

MacromoleculeName: Hyaluronan synthase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlorella virus
Molecular weightTheoretical: 65.337469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 CTYGDDRRLT 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 EHHHHHH

UniProtKB: Hyaluronan synthase

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Macromolecule #2: VNAR

MacromoleculeName: VNAR / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chiloscyllium plagiosum (whitespotted bambooshark)
Molecular weightTheoretical: 14.473181 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HHHHHHSSGL VPRGSGMLEV LFQGPQRVEQ TPTTTTKEAG ESLTINCVLR DSACALDNTY WYFTKKGATK KESLSNGGRY AETVNKASK SSSLRISDLR VEDSGTYHCK AYTAGIGCWN IFYEGGGTIL TVK

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Macromolecule #3: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE

MacromoleculeName: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE / type: ligand / ID: 3 / Number of copies: 1 / Formula: UD1
Molecular weightTheoretical: 607.354 Da
Chemical component information

ChemComp-UD1:
URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE

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Macromolecule #4: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 4 / Number of copies: 1 / 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: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI MORGAGNI
Image recordingFilm or detector model: FEI FALCON IV (4k 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: 1.8 µm / Nominal defocus min: 0.8 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7sp7

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 263915
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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