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- EMDB-40624: Chlorella virus Hyaluronan Synthase bound to GlcA extended GlcNAc... -

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Basic information

Entry
Database: EMDB / ID: EMD-40624
TitleChlorella virus Hyaluronan Synthase bound to GlcA extended GlcNAc primer and UDP
Map dataChlorella virus Hyaluronan Synthase bound to GlcA extended GlcNAc primer and UDP
Sample
  • Complex: Ternary complex of Chlorella virus hyaluronan synthase bound to nanobodies 872 and 886
    • Protein or peptide: Hyaluronan synthase
    • Protein or peptide: Nanobody 872
    • Protein or peptide: Nanobody 886
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
  • Ligand: MANGANESE (II) ION
Keywordshyaluronic acid / hyaluronan / HA / HAS / glycosyltransferase / GT / membrane protein / nanobody / n-acetylglucosamine / glucuronic acid / TRANSFERASE
Function / homologyhyaluronan synthase activity / Glycosyltransferase like family 2 / extracellular matrix assembly / hyaluronan biosynthetic process / : / Nucleotide-diphospho-sugar transferases / membrane / Hyaluronan synthase
Function and homology information
Biological speciesParamecium bursaria Chlorella virus 1 / Paramecium bursaria Chlorella virus CZ-2 / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsStephens Z / Zimmer J
Funding support United States, 2 items
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: 2024
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, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40624.png

Downloads & links

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Map

FileDownload / File: emd_40624.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChlorella virus Hyaluronan Synthase bound to GlcA extended GlcNAc primer and UDP
Projections & slices

Image control

Size
Brightness
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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

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.351
Minimum - Maximum-2.4263463 - 3.2774088
Average (Standard dev.)-0.00064665533 (±0.0616066)
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: Half Map 1

Fileemd_40624_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_40624_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Chlorella virus hyaluronan synthase bound to n...

EntireName: Ternary complex of Chlorella virus hyaluronan synthase bound to nanobodies 872 and 886
Components
  • Complex: Ternary complex of Chlorella virus hyaluronan synthase bound to nanobodies 872 and 886
    • Protein or peptide: Hyaluronan synthase
    • Protein or peptide: Nanobody 872
    • Protein or peptide: Nanobody 886
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Ternary complex of Chlorella virus hyaluronan synthase bound to n...

SupramoleculeName: Ternary complex of Chlorella virus hyaluronan synthase bound to nanobodies 872 and 886
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Paramecium bursaria Chlorella virus 1
Molecular weightTheoretical: 95.3 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: Paramecium bursaria Chlorella virus CZ-2
Molecular weightTheoretical: 65.890062 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 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 EHHHHHHHHH H

UniProtKB: Hyaluronan synthase

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Macromolecule #2: Nanobody 872

MacromoleculeName: Nanobody 872 / 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 K-12 (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLKV SCAASGRAFK TYRMAWFRQA PGKEREFVSG ISALETTYYA DSVKGRFTIS RDNTKNTVSL QMDSLKPED TAVYYCAARR YGGTDYTTTG SYDYWGQGTQ VTVSSHHHHH HEPEA

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Macromolecule #3: Nanobody 886

MacromoleculeName: Nanobody 886 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.737267 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
QVQLVESGGG SVQPGESLRL SCQASGRIVD VNDMAWYRQA PGKQRELVAR IARGGSTHYG DSAWGRFTIS RDNTRNTVYL QMTSLNVED TAVYYCNGEV KVGTRLSPFR TYWGRGTQVT VSSHHHHHHE PEA

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Macromolecule #5: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

ChemComp-UDP:
URIDINE-5'-DIPHOSPHATE / UDP*YM

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Macromolecule #6: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #7: 1,2-Distearoyl-sn-glycerophosphoethanolamine

MacromoleculeName: 1,2-Distearoyl-sn-glycerophosphoethanolamine / type: ligand / ID: 7 / Number of copies: 1 / Formula: 3PE
Molecular weightTheoretical: 748.065 Da
Chemical component information

ChemComp-3PE:
1,2-Distearoyl-sn-glycerophosphoethanolamine / phospholipid*YM

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Macromolecule #8: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 8 / 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

MicroscopeFEI TITAN 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 opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7sp6

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 220556
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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