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- PDB-8thj: Cryo-EM structure of the Tripartite ATP-independent Periplasmic (... -

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Basic information

Entry
Database: PDB / ID: 8thj
TitleCryo-EM structure of the Tripartite ATP-independent Periplasmic (TRAP) transporter SiaQM from Haemophilus influenzae (antiparallel dimer)
ComponentsSialic acid TRAP transporter permease protein SiaT
KeywordsTRANSPORT PROTEIN / Sugar transport / sialic acid / TRAP transporter / secondary active transport / ion transporter superfamily
Function / homology
Function and homology information


transmembrane transporter activity / carbohydrate transport / plasma membrane
Similarity search - Function
TRAP transporter large membrane protein DctM / TRAP transporter, small membrane protein DctQ / TRAP C4-dicarboxylate transport system permease DctM subunit / Tripartite ATP-independent periplasmic transporters, DctQ component / Tripartite ATP-independent periplasmic transporter, DctM component
Similarity search - Domain/homology
Chem-PGT / PHOSPHATIDYLETHANOLAMINE / Sialic acid TRAP transporter permease protein SiaT
Similarity search - Component
Biological speciesHaemophilus influenzae Rd KW20 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsDavies, J.S. / Currie, M.C. / Dobson, R.C.J. / North, R.A.
Funding support New Zealand, 2items
OrganizationGrant numberCountry
Marsden FundUOC1506 New Zealand
Ministry of Business, Innovation and Employment (New Zealand)UOCX1706 New Zealand
Citation
Journal: Elife / Year: 2024
Title: Structural and biophysical analysis of a tripartite ATP-independent periplasmic (TRAP) transporter.
Authors: Michael J Currie / James S Davies / Mariafrancesca Scalise / Ashutosh Gulati / Joshua D Wright / Michael C Newton-Vesty / Gayan S Abeysekera / Ramaswamy Subramanian / Weixiao Y Wahlgren / ...Authors: Michael J Currie / James S Davies / Mariafrancesca Scalise / Ashutosh Gulati / Joshua D Wright / Michael C Newton-Vesty / Gayan S Abeysekera / Ramaswamy Subramanian / Weixiao Y Wahlgren / Rosmarie Friemann / Jane R Allison / Peter D Mace / Michael D W Griffin / Borries Demeler / Soichi Wakatsuki / David Drew / Cesare Indiveri / Renwick C J Dobson / Rachel A North /
Abstract: Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or ...Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the -acetylneuraminate TRAP transporter (SiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous SiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the SiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity () for the complex between the soluble SiaP protein and SiaQM is in the micromolar range and that a related SiaP can bind SiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.
#1: Journal: Elife / Year: 2023
Title: Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
Authors: Currie, M.J. / Davies, J.S. / Scalise, M. / Gulati, A. / Wright, J.D. / Newton-Vesty, M.C. / Abeysekera, G.S. / Subramanian, R. / Wahlgren, W.Y. / Friemann, R. / Allison, J.R. / Mace, P.D. / ...Authors: Currie, M.J. / Davies, J.S. / Scalise, M. / Gulati, A. / Wright, J.D. / Newton-Vesty, M.C. / Abeysekera, G.S. / Subramanian, R. / Wahlgren, W.Y. / Friemann, R. / Allison, J.R. / Mace, P.D. / Griffin, M.D.W. / Demeler, B. / Wakatsuki, S. / Drew, D. / Indiveri, C. / Dobson, R.C.J. / North, R.A.
History
DepositionJul 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 22, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sialic acid TRAP transporter permease protein SiaT
B: Sialic acid TRAP transporter permease protein SiaT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,15610
Polymers144,0942
Non-polymers3,0628
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, equilibrium centrifugation, Sedimentation velocity analytical ultracentrifugation supports a dimeric state in solution
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sialic acid TRAP transporter permease protein SiaT / N-acetylneuraminic acid permease / N-acetylneuraminic acid transporter / Neu5Ac permease


Mass: 72046.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haemophilus influenzae Rd KW20 (bacteria)
Gene: siaT, siaQM, HI_0147 / Production host: Escherichia coli (E. coli) / Strain (production host): TOP10 / References: UniProt: P44543
#2: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE / Phosphatidylethanolamine


Mass: 734.039 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#4: 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.: 2 / Source method: obtained synthetically / Formula: C40H79O10P / Comment: phospholipid*YM
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: HiSiaQM transporter protein solubilised in amphipol A8-35
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Haemophilus influenzae Rd KW20 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: TOP10
Buffer solutionpH: 8
SpecimenConc.: 4.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm
Image recordingElectron dose: 73.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225044 / Symmetry type: POINT

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