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- EMDB-16364: The lipid linked oligosaccharide polymerase Wzy and its regulatin... -

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Basic information

Entry
Database: EMDB / ID: EMD-16364
TitleThe lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase Wzz form a complex in vivo and in vitro
Map dataWzzE:WzyE complex of Pectobacterium atrosepticum. Sharpened map
Sample
  • Complex: WzzE from Pectobacterium atrosepticum
    • Protein or peptide: ECA polysaccharide chain length modulation protein
Keywordsenterobacterial common antigen / lipopolysaccharide / Wzx/Wzy pathway / polysaccharide polymerase / polysaccharide co-polymerase / MEMBRANE PROTEIN
Function / homologyECA polysaccharide chain length modulation protein WzzE / enterobacterial common antigen biosynthetic process / Polysaccharide chain length determinant N-terminal domain / Chain length determinant protein / : / protein tyrosine kinase activity / plasma membrane / ECA polysaccharide chain length modulation protein
Function and homology information
Biological speciesPectobacterium atrosepticum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWeckener M / Woodward LS / Clarke BR / Liu H / Ward PN / Le Bas A / Bhella D / Whitfield C / Naismith JH
Funding support United Kingdom, Canada, 4 items
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, 2022-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16364.png

Downloads & links

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Map

FileDownload / File: emd_16364.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationWzzE:WzyE complex of Pectobacterium atrosepticum. Sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.12027797 - 0.18066095
Average (Standard dev.)0.000015709102 (±0.0059103332)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: WzzE:WzyE complex of Pectobacterium atrosepticum. DeepEMhancer processed map...

Fileemd_16364_additional_1.map
AnnotationWzzE:WzyE complex of Pectobacterium atrosepticum. DeepEMhancer processed map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: WzzE:WzyE complex of Pectobacterium atrosepticum. Half map 1

Fileemd_16364_half_map_1.map
AnnotationWzzE:WzyE complex of Pectobacterium atrosepticum. Half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: WzzE:WzyE complex of Pectobacterium atrosepticum. Half map 2

Fileemd_16364_half_map_2.map
AnnotationWzzE:WzyE complex of Pectobacterium atrosepticum. Half 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 : WzzE from Pectobacterium atrosepticum

EntireName: WzzE from Pectobacterium atrosepticum
Components
  • Complex: WzzE from Pectobacterium atrosepticum
    • Protein or peptide: ECA polysaccharide chain length modulation protein

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Supramolecule #1: WzzE from Pectobacterium atrosepticum

SupramoleculeName: WzzE from Pectobacterium atrosepticum / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Polysaccharide polymerase co-factor
Source (natural)Organism: Pectobacterium atrosepticum (bacteria)

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Macromolecule #1: ECA polysaccharide chain length modulation protein

MacromoleculeName: ECA polysaccharide chain length modulation protein / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium atrosepticum (bacteria)
Molecular weightTheoretical: 39.546781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMVKSENLS TGNALIDNEL DIRGLFRLLW RGKVWPIAIG LLFAVVALGY SYLVKQEWGA TSITDKPTVN MLGGYYSQQQ FLRNLDARS FSSPQPEQPS ISAGAYDEFI MQLAAYDTRR DFWLQTDYYK QRLEGDEKAD AALLDELVNN IQFTARDDGK K TNDSVKLV ...String:
GAMVKSENLS TGNALIDNEL DIRGLFRLLW RGKVWPIAIG LLFAVVALGY SYLVKQEWGA TSITDKPTVN MLGGYYSQQQ FLRNLDARS FSSPQPEQPS ISAGAYDEFI MQLAAYDTRR DFWLQTDYYK QRLEGDEKAD AALLDELVNN IQFTARDDGK K TNDSVKLV AETSSDSNTL LRQYVVFASQ RAANHLNEEI KGAWAARTIF MKSQIKRQEA VAKAIYDREV RSVELALKIA QQ QGISRSQ TDTPADEIPA SEMFLLGRPM LQARLETLQT SGPHYELDYD QNRAMLATLN VGPTLEASFQ TYRYLRTPEE PVK RDSPRR AFLMVMWGAI GVLVGAGVAL ARRLR

UniProtKB: ECA polysaccharide chain length modulation protein

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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: 8
Details: 50 mM phosphate buffer, pH 8, 200 mM NaCl, 0.026% (w/v) DDM
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Quantifoil R2/2 400 mesh grids were glow-discharged in the presence of pentyl-amine to drive protein into the ice..
DetailsThis membrane protein was purified in the presence of DDM

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2347 / Average exposure time: 8.5 sec. / Average electron dose: 85.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 464832
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Details: Relion 3.1 AutoRefine / Number images used: 190490
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe model was built into a density modified map - processed using DeepEMhancer
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8c0e:
The lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase Wzz form a complex in vivo and in vitro

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