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- PDB-8viw: Cryo-EM structure of heparosan synthase 2 from Pasteurella multoc... -

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Basic information

Entry
Database: PDB / ID: 8viw
TitleCryo-EM structure of heparosan synthase 2 from Pasteurella multocida with polysaccharide in the GlcNAc-T active site
ComponentsHeparosan synthase B
KeywordsTRANSFERASE / polysaccharide synthase / complex
Function / homologyGlycosyltransferase 2-like / Glycosyl transferase family 2 / Nucleotide-diphospho-sugar transferases / : / URIDINE-5'-DIPHOSPHATE / Heparosan synthase B
Function and homology information
Biological speciesPasteurella multocida (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKrahn, J.M. / Pedersen, L.C. / Liu, J. / Stancanelli, E. / Borgnia, M. / Vivarette, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIC ES102645 United States
CitationJournal: Acs Catalysis / Year: 2024
Title: Structural and Functional Analysis of Heparosan Synthase 2 from Pasteurella multocida to Improve the Synthesis of Heparin
Authors: Stancanelli, E. / Krahn, J.A. / Viverette, E. / Dutcher, R. / Pagadala, V. / Borgnia, M.J. / Liu, J. / Pedersen, L.C.
History
DepositionJan 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Heparosan synthase B
B: Heparosan synthase B
C: Heparosan synthase B
D: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)265,98124
Polymers258,1934
Non-polymers7,78820
Water724
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
Heparosan synthase B


Mass: 64548.277 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pasteurella multocida (bacteria) / Gene: hssB / Production host: Escherichia coli (E. coli) / References: UniProt: Q5SGE1
#2: Polysaccharide
beta-D-glucopyranuronic acid-(1-4)-2-deoxy-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D- ...beta-D-glucopyranuronic acid-(1-4)-2-deoxy-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranuronic acid-(1-4)-2-deoxy-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranuronic acid


Type: oligosaccharide / Mass: 1028.824 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpAb1-4DGlcpNSa1-4DGlcpAb1-4DGlcpNSa1-4DGlcpAb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,5,4/[a2122A-1b_1-5][a2122h-1a_1-5_2*NSO/3=O/3=O]/1-2-1-2-1/a4-b1_b4-c1_c4-d1_d4-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpA]{[(4+1)][a-D-GlcpNSO3]{[(4+1)][b-D-GlcpA]{[(4+1)][a-D-GlcpNSO3]{[(4+1)][b-D-GlcpA]{}}}}}LINUCSPDB-CARE
#3: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: pmHS2 with 7-mer polysaccharide / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pasteurella multocida (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 25 mM Tris pH 7.5, 87.5 mM NaCl, 1 mM MnCl2, 1 mM UDP and 1 mM NS-7mer (GlcA-GlcNS-GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP)
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: NITROGEN

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4544

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Processing

EM software
IDNameVersionCategory
11cryoSPARC4.3.1classification
12cryoSPARC4.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104255 / Algorithm: FOURIER SPACE / Num. of class averages: 169 / Symmetry type: POINT
RefinementCross valid method: NONE

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