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- PDB-7sp8: Chlorella virus Hyaluronan Synthase bound to UDP-GlcNAc -

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Basic information

Entry
Database: PDB / ID: 7sp8
TitleChlorella virus Hyaluronan Synthase bound to UDP-GlcNAc
Components
  • Hyaluronan synthase
  • Nanobody 872
  • Nanobody 881
KeywordsMEMBRANE PROTEIN / glycosyltransferase / hyaluronan
Function / homologyGlycosyltransferase like family 2 / Nucleotide-diphospho-sugar transferases / membrane / 1,2-Distearoyl-sn-glycerophosphoethanolamine / : / URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE / CHOLESTEROL HEMISUCCINATE / Hyaluronan synthase
Function and homology information
Biological speciesParamecium bursaria Chlorella virus CZ-2
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsMaloney, F.P. / Kuklewicz, J. / Zimmer, J.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 20, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Hyaluronan synthase
B: Nanobody 872
C: Nanobody 881
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,3388
Polymers95,3853
Non-polymers1,9525
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, TIRF photobleaching and co-purification experiments.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Hyaluronan synthase /


Mass: 65336.484 Da / Num. of mol.: 1 / Mutation: D302N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paramecium bursaria Chlorella virus CZ-2
Gene: CZ-2_118R, PBCVCZ2_118R / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C43 / References: UniProt: M1H2Q1

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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) / Plasmid: pMESy4 / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): WK6
#3: Antibody Nanobody 881


Mass: 15265.755 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pMESy4 / Production host: Escherichia coli K-12 (bacteria)

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Non-polymers , 4 types, 5 molecules

#4: Chemical ChemComp-UD1 / URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE


Mass: 607.354 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H27N3O17P2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-3PE / 1,2-Distearoyl-sn-glycerophosphoethanolamine / 3-SN-PHOSPHATIDYLETHANOLAMINE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / Phosphatidylethanolamine


Mass: 748.065 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H82NO8P / Comment: phospholipid*YM
#6: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#7: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Hyaluronan synthase in nanodiscs in complex with two camelid nanobodies.
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.0958 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Paramecium bursaria Chlorella virus CZ-21278251
31Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli BL21(DE3) (bacteria)469008C43pET28a
31Escherichia coli K-12 (bacteria)83333
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.1 MSodium ChlorideNaClSodium chloride1
20.02 MTris pH 7.5C4H11NO31
30.005 MBeta-mercaptoethanol2-MercaptoethanolC2H6OS1
40.005 MManganese ChlorideMnCl21
50.005 MUridine Diphosphate N-acetyl GlucosamineC17H27N3O17P21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Sample incubated on grid 30s before blotting. Blot 4 seconds with force 4.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8218 / Details: 40-frame movies were collected
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1cryoSPARC3.1particle selection
2EPU2.5.0.4799RELimage acquisition
4CTFFIND4.1.14CTF correction
10cryoSPARC3.1initial Euler assignment
11cryoSPARC3.1final Euler assignment
12cryoSPARC3.1classification
13cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4391341
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105056 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 66.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00146242
ELECTRON MICROSCOPYf_angle_d0.39688469
ELECTRON MICROSCOPYf_chiral_restr0.0386919
ELECTRON MICROSCOPYf_plane_restr0.00261041
ELECTRON MICROSCOPYf_dihedral_angle_d4.5776905

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