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- EMDB-13268: Streptococcus pneumoniae choline importer LicB in lipid nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-13268
TitleStreptococcus pneumoniae choline importer LicB in lipid nanodiscs
Map data
Sample
  • Complex: Choline importer in nanodiscs bound to nanobody
    • Protein or peptide: NanobodySingle-domain antibody
    • Protein or peptide: LicB protein
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
Function / homologyEamA domain / EamA-like transporter family / membrane => GO:0016020 / LicB protein
Function and homology information
Biological speciesStreptococcus pneumoniae TIGR4 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsPerez C / Baerland N
Funding support Switzerland, 1 items
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 AS / Cebrero G / Hutter CA / Seeger MA / Veening JW / Perez C
History
DepositionJul 29, 2021-
Header (metadata) releaseMar 16, 2022-
Map releaseMar 16, 2022-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.135
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.135
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7paf
  • Surface level: 0.135
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13268.png

Downloads & links

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Map

FileDownload / File: emd_13268.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.878 Å
Density
Contour LevelBy AUTHOR: 0.135 / Movie #1: 0.135
Minimum - Maximum-0.6427229 - 0.95486265
Average (Standard dev.)-4.6419427e-05 (±0.01275228)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 337.152 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8780.8780.878
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z337.152337.152337.152
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.6430.955-0.000

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Supplemental data

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Mask #1

Fileemd_13268_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Choline importer in nanodiscs bound to nanobody

EntireName: Choline importer in nanodiscs bound to nanobody
Components
  • Complex: Choline importer in nanodiscs bound to nanobody
    • Protein or peptide: NanobodySingle-domain antibody
    • Protein or peptide: LicB protein
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE

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Supramolecule #1: Choline importer in nanodiscs bound to nanobody

SupramoleculeName: Choline importer in nanodiscs bound to nanobody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Streptococcus pneumoniae TIGR4 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightExperimental: 260 KDa

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Macromolecule #1: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.802365 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
GSSSQVQLVE SGGGSVQAGG SLRLSCAASG TIHAIGYLGW FRQAPGKERE GVAALTTYDG WTYYADSVKG RFTVSLDNAK NTVYLQMNS LKPEDTALYY CAAADDGWMF PLYHNHYEYW GQGTQVTVSA GRAGEQKLIS EEDLNSAVDH HHHHH

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Macromolecule #2: LicB protein

MacromoleculeName: LicB protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pneumoniae TIGR4 (bacteria) / Strain: ATCC BAA-334 / TIGR4
Molecular weightTheoretical: 31.647631 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKSKNGVPFG LLSGIFWGLG LTVSAYIFSI FTDLSPFVVA ATHDFLSIFI LLAFLLVKEG KVRLSIFLNI RNVSVIIGAL LAGPIGMQA NLYAVKYIGS SLASSVSAIY PAISVLLAFF FLKHKISKNT VFGIVLIIGG IIAQTYKVEQ VNSFYIGILC A LVCAIAWG ...String:
MKSKNGVPFG LLSGIFWGLG LTVSAYIFSI FTDLSPFVVA ATHDFLSIFI LLAFLLVKEG KVRLSIFLNI RNVSVIIGAL LAGPIGMQA NLYAVKYIGS SLASSVSAIY PAISVLLAFF FLKHKISKNT VFGIVLIIGG IIAQTYKVEQ VNSFYIGILC A LVCAIAWG SESVLSSFAM ESELSEIEAL LIRQVTSFLS YLVIVLFSHQ SFTAVANGQL LGLMIVFAAF DMISYLAYYI AI NRLQPAK ATGLNVSYVV WTVLFAVVFL GAPLDMLTIM TSLVVIAGVY IIIKE

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Macromolecule #3: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 3 / Number of copies: 1 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM / Phosphatidylglycerol

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. v3.2.0)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.2.0)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. v3.2.0)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v3.2.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2.0) / Number images used: 78649

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

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

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