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- EMDB-25370: Chlorella virus Hyaluronan Synthase in the GlcNAc-primed, channel... -

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Basic information

Entry
Database: EMDB / ID: EMD-25370
TitleChlorella virus Hyaluronan Synthase in the GlcNAc-primed, channel-open state
Map data
Sample
  • Complex: Hyaluronan synthase in nanodiscs in complex with two camelid nanobodies.
    • Protein or peptide: Nanobody 872
    • Protein or peptide: Nanobody 881
    • Protein or peptide: Hyaluronan synthase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
Function / homologyGlycosyltransferase like family 2 / Nucleotide-diphospho-sugar transferases / membrane / Hyaluronan synthase
Function and homology information
Biological speciesParamecium bursaria Chlorella virus CZ-2 / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMaloney FP / Kuklewicz J / Zimmer J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21AI148853 United States
CitationJournal: Nature / Year: 2022
Title: Structure, substrate recognition and initiation of hyaluronan synthase.
Authors: Finn P Maloney / Jeremi Kuklewicz / Robin A Corey / Yunchen Bi / Ruoya Ho / Lukasz Mateusiak / Els Pardon / Jan Steyaert / Phillip J Stansfeld / Jochen Zimmer /
Abstract: Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix. The high- ...Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.
History
DepositionNov 2, 2021-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateApr 20, 2022-
Current statusApr 20, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25370.png

Downloads & links

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Map

FileDownload / File: emd_25370.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 6.84
Minimum - Maximum-11.039706 - 34.852234
Average (Standard dev.)-0.00081937836 (±0.8067743)
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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Sample components

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Entire : Hyaluronan synthase in nanodiscs in complex with two camelid nano...

EntireName: Hyaluronan synthase in nanodiscs in complex with two camelid nanobodies.
Components
  • Complex: Hyaluronan synthase in nanodiscs in complex with two camelid nanobodies.
    • Protein or peptide: Nanobody 872
    • Protein or peptide: Nanobody 881
    • Protein or peptide: Hyaluronan synthase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine

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Supramolecule #1: Hyaluronan synthase in nanodiscs in complex with two camelid nano...

SupramoleculeName: Hyaluronan synthase in nanodiscs in complex with two camelid nanobodies.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Paramecium bursaria Chlorella virus CZ-2
Molecular weightTheoretical: 95.8 KDa

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Macromolecule #1: Nanobody 872

MacromoleculeName: Nanobody 872 / type: protein_or_peptide / ID: 1 / 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 #2: Nanobody 881

MacromoleculeName: Nanobody 881 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 15.265755 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLRL ACAASGRIFS SDTLAWFRRA PGKEREFVAA SRWSGGGTDY ADSVKGRFTF SRDNTRNTMC LEMNSLKPE DTAVYYCALR TARDSYYYTR NPTGYDYWGQ GTQVTVSSHH HHHHEPEA

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Macromolecule #3: Hyaluronan synthase

MacromoleculeName: Hyaluronan synthase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Paramecium bursaria Chlorella virus CZ-2
Molecular weightTheoretical: 65.336484 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 CTYGDNRRLT 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

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: 1,2-Distearoyl-sn-glycerophosphoethanolamine

MacromoleculeName: 1,2-Distearoyl-sn-glycerophosphoethanolamine / type: ligand / ID: 6 / Number of copies: 1 / Formula: 3PE
Molecular weightTheoretical: 748.065 Da
Chemical component information

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

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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
Component:
ConcentrationFormulaName
0.1 MNaClSodium chlorideSodium Chloride
0.02 MC4H11NO3Tris pH 7.5
0.005 MC2H6OSBeta-mercaptoethanol2-Mercaptoethanol
0.005 MMnCl2Manganese Chloride
0.005 MC17H27N3O17P2Uridine Diphosphate N-acetyl Glucosamine
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
Details: Sample incubated on grid 30s before blotting. Blot 4 seconds with force 4.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8218 / Average electron dose: 51.0 e/Å2 / Details: 40-frame movies were collected
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4391341
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14)
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.1)
Details: Discrete biochemical states were separated during 3D variability analysis and sorting based on a high resolution reconstruction. Classes belonging to each state were pooled and subjected to ...Details: Discrete biochemical states were separated during 3D variability analysis and sorting based on a high resolution reconstruction. Classes belonging to each state were pooled and subjected to a final round of masked 3D refinement.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 104995
FSC plot (resolution estimation)

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