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- PDB-8y4x: Apo form of Tripartite ATP-independent Periplasmic (TRAP) transpo... -

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Basic information

Entry
Database: PDB / ID: 8y4x
TitleApo form of Tripartite ATP-independent Periplasmic (TRAP) transporter from Fusobacterium nucleatum.
Components
  • N-acetylneuraminate transporter small subunit
  • Nanobody against FnTRAP
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / sialic acid transporter / TRAP transporter / MEMBRANE PROTEIN / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homologyTRAP transporter large membrane protein DctM / TRAP C4-dicarboxylate transport system permease DctM subunit / : / Tripartite ATP-independent periplasmic transporters, DctQ component / Tripartite ATP-independent periplasmic transporter, DctM component / transmembrane transporter activity / plasma membrane / PHOSPHATIDYLETHANOLAMINE / N-acetylneuraminate transporter small subunit
Function and homology information
Biological speciesFusobacterium nucleatum (bacteria)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGoyal, P. / Ramaswamy, S. / Vinothkumar, K.R.
Funding support India, 2items
OrganizationGrant numberCountry
Other governmentIA/E/16/1/502999
Department of Biotechnology (DBT, India)DBT/PR/12422/MED/31/287/2014 India
CitationJournal: Elife / Year: 2025
Title: Molecular determinants of Neu5Ac binding to a tripartite ATP independent periplasmic (TRAP) transporter.
Authors: Parveen Goyal / KanagaVijayan Dhanabalan / Mariafrancesca Scalise / Rosmarie Friemann / Cesare Indiveri / Renwick C J Dobson / Kutti R Vinothkumar / Subramanian Ramaswamy /
Abstract: -Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and ... -Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na binding sites. Two of the Na-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.
History
DepositionJan 31, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: N-acetylneuraminate transporter small subunit
B: Nanobody against FnTRAP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,3885
Polymers88,6082
Non-polymers7803
Water00
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 N-acetylneuraminate transporter small subunit


Mass: 74198.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Fusobacterium nucleatum (bacteria) / Gene: FN1473 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8RDN8
#2: Antibody Nanobody against FnTRAP


Mass: 14408.753 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
Has ligand of interestN
Has protein modificationY

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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: Apo form of TRAP transporter in 1:1 complex with its nanobody.
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Fusobacterium nucleatum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 2.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: OTHER
Electron lensMode: OTHER / Nominal magnification: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 27.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 960 / Details: 941 exposures were used.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM softwareName: PHENIX / Version: 1.21_5207: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 385668
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141272 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 42.52 / Protocol: OTHER / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0055939
ELECTRON MICROSCOPYf_angle_d0.528042
ELECTRON MICROSCOPYf_dihedral_angle_d12.0622133
ELECTRON MICROSCOPYf_chiral_restr0.041939
ELECTRON MICROSCOPYf_plane_restr0.005970

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