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- PDB-7paf: Streptococcus pneumoniae choline importer LicB in lipid nanodiscs -

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Basic information

Entry
Database: PDB / ID: 7paf
TitleStreptococcus pneumoniae choline importer LicB in lipid nanodiscs
Components
  • LicB protein
  • NanobodySingle-domain antibody
KeywordsTRANSPORT PROTEIN / Importer / Choline / nanodisc / Nanobody
Function / homologyEamA domain / EamA-like transporter family / membrane => GO:0016020 / Chem-PGT / LicB protein
Function and homology information
Biological speciessynthetic construct (others)
Streptococcus pneumoniae TIGR4 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsPerez, C. / Baerland, N.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationPP00P3_170607 Switzerland
Citation
Journal: Sci Adv / Year: 2022
Title: Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification.
Authors: Natalie Bärland / Anne-Stéphanie Rueff / Gonzalo Cebrero / Cedric A J Hutter / Markus A Seeger / Jan-Willem Veening / Camilo Perez /
Abstract: Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. ...Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo-electron microscopy and crystal structures of LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.
#1: Journal: Biorxiv / Year: 2021
Title: Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification
Authors: Barland, N. / Rueff, A.S. / Cebrero, G. / Hutter, C.A. / Seeger, M.A. / Veening, J.W. / Perez, C.
History
DepositionJul 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 16, 2022Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: Nanobody
A: LicB protein
C: Nanobody
D: LicB protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,6515
Polymers96,9004
Non-polymers7511
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3150 Å2
ΔGint-36 kcal/mol
Surface area37860 Å2

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Components

#1: Antibody Nanobody / Single-domain antibody


Mass: 16802.365 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)
#2: Protein LicB protein


Mass: 31647.631 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae TIGR4 (bacteria)
Strain: ATCC BAA-334 / TIGR4 / Gene: licB, SP_1268 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0H2UQH5
#3: Chemical ChemComp-PGT / (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / PHOSPHATIDYLGLYCEROL / 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)](SODIUM SALT) / Phosphatidylglycerol


Mass: 751.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H79O10P / Comment: phospholipid*YM
Has ligand of interestN

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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: Choline importer in nanodiscs bound to nanobody / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.260 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pneumoniae TIGR4 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 0 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCv3.2.0particle selection
4cryoSPARCv3.2.0CTF correction
10cryoSPARCv3.2.0initial Euler assignment
11cryoSPARCv3.2.0final Euler assignment
12cryoSPARCv3.2.0classification
13cryoSPARCv3.2.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78649 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036454
ELECTRON MICROSCOPYf_angle_d0.5598788
ELECTRON MICROSCOPYf_dihedral_angle_d8.8933684
ELECTRON MICROSCOPYf_chiral_restr0.041042
ELECTRON MICROSCOPYf_plane_restr0.0041070

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