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- PDB-8va1: S. aureus TarL H300N in complex with CDP-ribitol (single tetramer) -

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Basic information

Entry
Database: PDB / ID: 8va1
TitleS. aureus TarL H300N in complex with CDP-ribitol (single tetramer)
ComponentsTeichoic acid ribitol-phosphate polymerase TarL
KeywordsTRANSFERASE / glycosyltransferase / polymerase / monotopic / amphipathic
Function / homology
Function and homology information


CDP-ribitol ribitolphosphotransferase / CDP-ribitol ribitolphosphotransferase activity / CDP-glycerol glycerophosphotransferase activity / teichoic acid biosynthetic process / cell wall organization / plasma membrane
Similarity search - Function
: / CDP-glycerol glycerophosphotransferase / CDP-glycerol glycerophosphotransferase, C-terminal domain / CDP-glycerol glycerophosphotransferase, N-terminal domain / CDP-Glycerol:Poly(glycerophosphate) glycerophosphotransferase
Similarity search - Domain/homology
CDP-ribitol / Teichoic acid ribitol-phosphate polymerase TarL
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLi, F.K.K. / Worrall, L.J. / Strynadka, N.C.J.
Funding support Canada, United States, 4items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canadian Institutes of Health Research (CIHR) Canada
Howard Hughes Medical Institute (HHMI) United States
Canada Research Chairs Canada
CitationJournal: Sci Adv / Year: 2024
Title: Cryo-EM analysis of TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance.
Authors: Franco K K Li / Liam J Worrall / Robert T Gale / Eric D Brown / Natalie C J Strynadka /
Abstract: Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of ...Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo-electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate-ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.
History
DepositionDec 10, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Teichoic acid ribitol-phosphate polymerase TarL
B: Teichoic acid ribitol-phosphate polymerase TarL
C: Teichoic acid ribitol-phosphate polymerase TarL
D: Teichoic acid ribitol-phosphate polymerase TarL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,1928
Polymers274,0434
Non-polymers2,1494
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
Teichoic acid ribitol-phosphate polymerase TarL / Poly(ribitol phosphate) polymerase TarL / Ribitol-phosphate polymerase TarL / Tar polymerase TarL


Mass: 68510.703 Da / Num. of mol.: 4 / Mutation: H300N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: tarL, SAOUHSC_00227 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q2G1B8, CDP-ribitol ribitolphosphotransferase
#2: Chemical
ChemComp-V2V / CDP-ribitol / [(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl (2R,3S,4S)-2,3,4,5-tetrahydroxypentyl dihydrogen diphosphate


Mass: 537.307 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H25N3O15P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: S. aureus TarL H300N with CDP-ribitol and CHAPS / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2500 mMSodium chloride1
36.5 mMCHAPS1
SpecimenConc.: 9.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
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 FIELDBright-field microscopy / Calibrated magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 19380

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
9RELION3.1initial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 190753 / Symmetry type: POINT
Atomic model buildingPDB-ID: 3L7L

3l7l


Accession code: 3L7L / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.57 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004219425
ELECTRON MICROSCOPYf_angle_d0.671126294
ELECTRON MICROSCOPYf_chiral_restr0.04462834
ELECTRON MICROSCOPYf_plane_restr0.00543335
ELECTRON MICROSCOPYf_dihedral_angle_d7.97922547

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