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- PDB-8c0e: The lipid linked oligosaccharide polymerase Wzy and its regulatin... -

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Basic information

Entry
Database: PDB / ID: 8c0e
TitleThe lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase Wzz form a complex in vivo and in vitro
ComponentsECA polysaccharide chain length modulation protein
KeywordsMEMBRANE PROTEIN / enterobacterial common antigen / lipopolysaccharide / Wzx/Wzy pathway / polysaccharide polymerase / polysaccharide co-polymerase
Function / homologyECA polysaccharide chain length modulation protein WzzE / enterobacterial common antigen biosynthetic process / Polysaccharide chain length determinant N-terminal domain / Chain length determinant protein / : / plasma membrane / ECA polysaccharide chain length modulation protein
Function and homology information
Biological speciesPectobacterium atrosepticum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWeckener, M. / Woodward, L.S. / Clarke, B.R. / Liu, H. / Ward, P.N. / Le Bas, A. / Bhella, D. / Whitfield, C. / Naismith, J.H.
Funding support United Kingdom, Canada, 4items
OrganizationGrant numberCountry
Wellcome Trust20289/Z/16/Z United Kingdom
Wellcome Trust100209/Z/12/Z United Kingdom
Canadian Institutes of Health Research (CIHR)FDN-2016-148364 Canada
Medical Research Council (MRC, United Kingdom)MC_UU_12014/7 United Kingdom
CitationJournal: Open Biol / Year: 2023
Title: The lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase, Wzz, from enterobacterial common antigen biosynthesis form a complex.
Authors: Miriam Weckener / Laura S Woodward / Bradley R Clarke / Huanting Liu / Philip N Ward / Audrey Le Bas / David Bhella / Chris Whitfield / James H Naismith /
Abstract: The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the . ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of ...The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the . ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from form a complex , and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.
History
DepositionDec 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ECA polysaccharide chain length modulation protein
B: ECA polysaccharide chain length modulation protein
C: ECA polysaccharide chain length modulation protein
D: ECA polysaccharide chain length modulation protein
E: ECA polysaccharide chain length modulation protein
F: ECA polysaccharide chain length modulation protein
G: ECA polysaccharide chain length modulation protein
H: ECA polysaccharide chain length modulation protein


Theoretical massNumber of molelcules
Total (without water)316,3748
Polymers316,3748
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ECA polysaccharide chain length modulation protein


Mass: 39546.781 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum (bacteria) / Gene: wzzE / Production host: Escherichia coli (E. coli) / References: UniProt: Q6CZE3

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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: WzzE from Pectobacterium atrosepticum / Type: COMPLEX / Details: Polysaccharide polymerase co-factor / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pectobacterium atrosepticum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43
Buffer solutionpH: 8
Details: 50 mM phosphate buffer, pH 8, 200 mM NaCl, 0.026% (w/v) DDM
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This membrane protein was purified in the presence of DDM
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Quantifoil R2/2 400 mesh grids were glow-discharged in the presence of pentyl-amine to drive protein into the ice.

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.5 sec. / Electron dose: 85 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2347
Image scansMovie frames/image: 20

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Processing

SoftwareName: PHENIX / Version: dev_4788: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 464832
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 190490 / Algorithm: FOURIER SPACE / Details: Relion 3.1 AutoRefine / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: The model was built into a density modified map - processed using DeepEMhancer
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00418737
ELECTRON MICROSCOPYf_angle_d0.70225359
ELECTRON MICROSCOPYf_dihedral_angle_d4.582564
ELECTRON MICROSCOPYf_chiral_restr0.0392771
ELECTRON MICROSCOPYf_plane_restr0.0083323

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