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- PDB-6rbg: full-length bacterial polysaccharide co-polymerase -

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Basic information

Entry
Database: PDB / ID: 6rbg
Titlefull-length bacterial polysaccharide co-polymerase
ComponentsChain length determinant protein
KeywordsMEMBRANE PROTEIN / enzyme / polysaccharide / co-polymerase
Function / homologyPolysaccharide chain length determinant N-terminal domain / Chain length determinant protein / lipopolysaccharide biosynthetic process / protein tyrosine kinase activity / membrane => GO:0016020 / plasma membrane / Chain length determinant protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsWiseman, B. / Nitharwal, R.G. / Hogbom, M.
Funding support Sweden, 2items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation Sweden
Swedish Research Council Sweden
CitationJournal: Nat Commun / Year: 2021
Title: Structure of a full-length bacterial polysaccharide co-polymerase.
Authors: Benjamin Wiseman / Ram Gopal Nitharwal / Göran Widmalm / Martin Högbom /
Abstract: Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of ...Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of the most widely disseminated pathways of polysaccharide biosynthesis is the inner membrane bound Wzy-dependent pathway. Here we present the 3.0 Å structure of the co-polymerase component of this pathway, WzzB from E. coli solved by single-particle cryo-electron microscopy. The overall architecture is octameric and resembles a box jellyfish containing a large bell-shaped periplasmic domain with the 2-helix transmembrane domain from each protomer, positioned 32 Å apart, encircling a large empty transmembrane chamber. This structure also reveals the architecture of the transmembrane domain, including the location of key residues for the Wzz-family of proteins and the Wzy-dependent pathway present in many Gram-negative bacteria, explaining several of the previous biochemical and mutational studies and lays the foundation for future investigations.
History
DepositionApr 10, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Jan 27, 2021Group: Database references / Category: citation / citation_author
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
Revision 1.3May 22, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Chain length determinant protein
B: Chain length determinant protein
C: Chain length determinant protein
D: Chain length determinant protein
E: Chain length determinant protein
F: Chain length determinant protein
G: Chain length determinant protein
H: Chain length determinant protein


Theoretical massNumber of molelcules
Total (without water)306,9658
Polymers306,9658
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis, gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36180 Å2
ΔGint-130 kcal/mol
Surface area130210 Å2
MethodPISA

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Components

#1: Protein
Chain length determinant protein / Polysaccharide antigen chain regulator


Mass: 38370.621 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: wzzB, cld, rol, wzz, b2027, JW5836 / Production host: Escherichia coli DH5[alpha] (bacteria) / Variant (production host): C41 / References: UniProt: P76372

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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: Octameric bacterial polysaccharide co-polymerase complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli DH5[alpha] (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 40 seconds at 20 mA / Grid material: COPPER / Grid type: Quantifoil R2/2
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 / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2347
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9cryoSPARC2initial Euler assignment
10cryoSPARC2final Euler assignment
11cryoSPARC2classification
12cryoSPARC23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 90297
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52378 / Symmetry type: POINT
Atomic model buildingB value: 87 / Protocol: FLEXIBLE FIT / Space: REAL

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