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- PDB-8p3p: Full-length bacterial polysaccharide co-polymerase WzzE mutant R2... -

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Basic information

Entry
Database: PDB / ID: 8p3p
TitleFull-length bacterial polysaccharide co-polymerase WzzE mutant R267E from E. coli. C4 symmetry
ComponentsECA polysaccharide chain length modulation protein
KeywordsMEMBRANE PROTEIN / Complex / Lipopolysaccharide / bacterial 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 / protein tyrosine kinase activity / plasma membrane / ECA polysaccharide chain length modulation protein
Function and homology information
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsWiseman, B. / Hogbom, M.
Funding support Sweden, 3items
OrganizationGrant numberCountry
Swedish Research Council2017-04018 Sweden
Knut and Alice Wallenberg Foundation2017.0275 Sweden
Knut and Alice Wallenberg Foundation2019.0436 Sweden
CitationJournal: Commun Biol / Year: 2023
Title: Alternating L4 loop architecture of the bacterial polysaccharide co-polymerase WzzE.
Authors: Benjamin Wiseman / Göran Widmalm / Martin Högbom /
Abstract: Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often ...Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often polymerized by the inner membrane bound Wzy-dependent pathway. By employing cryo-electron microscopy we show that WzzE, the co-polymerase component of this pathway that is responsible for the length modulation of the enterobacterial common antigen, is octameric with alternating up-down conformations of its L4 loops. The alternating up-down nature of these essential loops, located at the top of the periplasmic bell, are modulated by clashing helical faces between adjacent protomers that flank the L4 loops around the octameric periplasmic bell. This alternating arrangement and a highly negatively charged binding face create a dynamic environment in which the polysaccharide chain is extended, and suggest a ratchet-type mechanism for polysaccharide elongation.
History
DepositionMay 18, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 9, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: ECA polysaccharide chain length modulation protein
H: ECA polysaccharide chain length modulation protein
A: ECA polysaccharide chain length modulation protein
B: 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


Theoretical massNumber of molelcules
Total (without water)331,0648
Polymers331,0648
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ECA polysaccharide chain length modulation protein


Mass: 41382.941 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: wzzE, wzz, yifC, b3785, JW5601 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41 / References: UniProt: P0AG00

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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: Bacterial Polysaccharide Co-polymerase WzzE / Type: COMPLEX / Details: Homo octameric complex / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.32 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41
Buffer solutionpH: 8
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11932

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2EPUimage acquisition
4cryoSPARC3CTF correction
7PHENIXmodel fitting
9PHENIX1.20.1_4487:model refinement
10cryoSPARC3initial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARC3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2349935
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 308817 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 125 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 8BHW

8bhw


Accession code: 8BHW / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00321648
ELECTRON MICROSCOPYf_angle_d0.61829296
ELECTRON MICROSCOPYf_dihedral_angle_d3.4472952
ELECTRON MICROSCOPYf_chiral_restr0.0353184
ELECTRON MICROSCOPYf_plane_restr0.0043840

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