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- EMDB-38926: Apo form of Tripartite ATP-independent Periplasmic (TRAP) transpo... -

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Basic information

Entry
Database: EMDB / ID: EMD-38926
TitleApo form of Tripartite ATP-independent Periplasmic (TRAP) transporter from Fusobacterium nucleatum.
Map dataextracted map
Sample
  • Complex: Apo form of TRAP transporter in 1:1 complex with its nanobody.
    • Protein or peptide: N-acetylneuraminate transporter small subunit
    • Protein or peptide: Nanobody against FnTRAP
  • Ligand: SODIUM ION
  • Ligand: PHOSPHATIDYLETHANOLAMINE
Keywordssialic 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 / N-acetylneuraminate transporter small subunit
Function and homology information
Biological speciesFusobacterium nucleatum (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGoyal P / Ramaswamy S / Vinothkumar KR
Funding support India, 2 items
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, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38926.png

Downloads & links

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Map

FileDownload / File: emd_38926.map.gz / Format: CCP4 / Size: 4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationextracted map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.07 Å/pix.
x 92 pix.
= 98.44 Å		1.07 Å/pix.
x 116 pix.
= 124.12 Å		1.07 Å/pix.
x 99 pix.
= 105.93 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.1
Minimum - Maximum-5.238249 - 8.707461
Average (Standard dev.)0.078333616 (±0.5320616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin97109109
Dimensions1169992
Spacing9211699
CellA: 98.44 Å / B: 124.12 Å / C: 105.93001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Unextracted half map. No mask applied

Fileemd_38926_half_map_1.map
AnnotationUnextracted half map. No mask applied
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unextracted half map. No mask applied.

Fileemd_38926_half_map_2.map
AnnotationUnextracted half map. No mask applied.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Apo form of TRAP transporter in 1:1 complex with its nanobody.

EntireName: Apo form of TRAP transporter in 1:1 complex with its nanobody.
Components
  • Complex: Apo form of TRAP transporter in 1:1 complex with its nanobody.
    • Protein or peptide: N-acetylneuraminate transporter small subunit
    • Protein or peptide: Nanobody against FnTRAP
  • Ligand: SODIUM ION
  • Ligand: PHOSPHATIDYLETHANOLAMINE

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Supramolecule #1: Apo form of TRAP transporter in 1:1 complex with its nanobody.

SupramoleculeName: Apo form of TRAP transporter in 1:1 complex with its nanobody.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Fusobacterium nucleatum (bacteria)

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Macromolecule #1: N-acetylneuraminate transporter small subunit

MacromoleculeName: N-acetylneuraminate transporter small subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Fusobacterium nucleatum (bacteria)
Molecular weightTheoretical: 74.198938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGGSHHHHHH GSWSHPQFEK ASMTGGQQMG RDLYDDDDKD RWGSELEMKV FNKLEEWLGG SLFIGMFVIL VMQIFSRQIF NSPLIWSEE LSRLIFVYVG LLGVSMGIRS QQHIMIDFLY AKFPKSMQKI IFTIIQILIL ACLIFFLYFG YDLFIKKEEI E IVSLGISM ...String:
MGGSHHHHHH GSWSHPQFEK ASMTGGQQMG RDLYDDDDKD RWGSELEMKV FNKLEEWLGG SLFIGMFVIL VMQIFSRQIF NSPLIWSEE LSRLIFVYVG LLGVSMGIRS QQHIMIDFLY AKFPKSMQKI IFTIIQILIL ACLIFFLYFG YDLFIKKEEI E IVSLGISM KWMYLALPLI TLLMLVRFYQ AYSENYAQNK VYIKPIFILA LMIILVLIAF IKPELFKILK LSNYFDLGEM TI YYVLIAW LVMIFFGVPV GWSLLVACIL YFALTRWKVV YFAADKLVYS LDSFSLLSVP FFILTGILMN GAGITERIFN FAK AMLGHY TGGMGHVNVA ASLIFSGMSG SAIADAGGLG QLEIKAMRDE GYDDDICGGL TAASCIIGPL VPPSISMIIY GVIA NQSIA KLFLAGFVPG FLTTIALMIM NYFVCKKRGY KKTAKASPKE RWIAFKKSFW ALLTPILIIG GIFSGIFTPT EAAVI ATFY SIILGGFIYK ELTVKSFFKH CVEAVAISGV TVLMIMTVTF FGDIIAREQV AMRVAEIFIK YATSPMMVLV MINLLL LFL GMFIDALALQ FLVLPMLIPI AEQVGIDLVF FGVMTTLNMM IGILTPPMGM ALFVVAQVGK MSVSTVAKGV LPFLLPI FI TLVIITIFPQ IILFLPNLIV GG

UniProtKB: N-acetylneuraminate transporter small subunit

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Macromolecule #2: Nanobody against FnTRAP

MacromoleculeName: Nanobody against FnTRAP / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 14.408753 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCTTSGFNFD DYAIGWFRQA PGKEREGVSC IHCTAYTPYY ARSVRDRFTI SSDNATNTVF LQMNNLRPE DTAVYYCVAD ATRYPYPEFY DYVGQGTQVT VSSHHHHHH

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 2
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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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

Concentration2.9 mg/mL
BufferpH: 8
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 960 / Average exposure time: 60.0 sec. / Average electron dose: 27.7 e/Å2 / Details: 941 exposures were used.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130841 / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 385668
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio in cryosparc
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 141272
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 64 / Avg.num./class: 2207
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 42.52
Output model

PDB-8y4x:
Apo form of Tripartite ATP-independent Periplasmic (TRAP) transporter from Fusobacterium nucleatum.

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