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- PDB-8t53: S. enterica WbaP in a styrene maleic acid liponanoparticle -

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Basic information

Entry
Database: PDB / ID: 8t53
TitleS. enterica WbaP in a styrene maleic acid liponanoparticle
ComponentsUndecaprenyl-phosphate galactose phosphotransferase
KeywordsTRANSFERASE / phosphoglycosyl transferase / liponanopaticle / SMALP / O-antigen / Salmonella enterica
Function / homology
Function and homology information


undecaprenyl-phosphate galactose phosphotransferase / undecaprenyl-phosphate galactose phosphotransferase activity / phosphotransferase activity, for other substituted phosphate groups / O antigen biosynthetic process / plasma membrane
Similarity search - Function
Undecaprenyl-phosphate galactose phosphotransferase, WbaP / Exopolysaccharide biosynthesis polyprenyl glycosylphosphotransferase / CoA-binding domain / Bacterial sugar transferase / Bacterial sugar transferase
Similarity search - Domain/homology
Undecaprenyl-phosphate galactose phosphotransferase
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsDodge, G.J. / Imperiali, B.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM134576 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131627 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM039334 United States
CitationJournal: Elife / Year: 2024
Title: Mapping the architecture of the initiating phosphoglycosyl transferase from O-antigen biosynthesis in a liponanoparticle.
Authors: Greg J Dodge / Alyssa J Anderson / Yi He / Weijing Liu / Rosa Viner / Barbara Imperiali /
Abstract: Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have ...Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.
History
DepositionJun 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Undecaprenyl-phosphate galactose phosphotransferase
B: Undecaprenyl-phosphate galactose phosphotransferase


Theoretical massNumber of molelcules
Total (without water)122,7652
Polymers122,7652
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Undecaprenyl-phosphate galactose phosphotransferase / / Galactosyl-P-P-undecaprenol synthase / WbaP


Mass: 61382.699 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Gene: rfbP, STM2082 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43
References: UniProt: P26406, undecaprenyl-phosphate galactose phosphotransferase

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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: WbaP dimer embedded in a styrene maleic acid liponanoparticle
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: YES
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.13 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4841

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487:model refinement
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1266538
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196663 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026848
ELECTRON MICROSCOPYf_angle_d0.5579278
ELECTRON MICROSCOPYf_dihedral_angle_d4.451872
ELECTRON MICROSCOPYf_chiral_restr0.0411024
ELECTRON MICROSCOPYf_plane_restr0.0051122

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