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Yorodumi- PDB-8thj: Cryo-EM structure of the Tripartite ATP-independent Periplasmic (... -
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-Basic information
Entry | Database: PDB / ID: 8thj | |||||||||
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Title | Cryo-EM structure of the Tripartite ATP-independent Periplasmic (TRAP) transporter SiaQM from Haemophilus influenzae (antiparallel dimer) | |||||||||
Components | Sialic acid TRAP transporter permease protein SiaT | |||||||||
Keywords | TRANSPORT PROTEIN / Sugar transport / sialic acid / TRAP transporter / secondary active transport / ion transporter superfamily | |||||||||
Function / homology | Function and homology information | |||||||||
Biological species | Haemophilus influenzae Rd KW20 (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å | |||||||||
Authors | Davies, J.S. / Currie, M.C. / Dobson, R.C.J. / North, R.A. | |||||||||
Funding support | New Zealand, 2items
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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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8thj.cif.gz | 247.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8thj.ent.gz | 195.8 KB | Display | PDB format |
PDBx/mmJSON format | 8thj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/th/8thj ftp://data.pdbj.org/pub/pdb/validation_reports/th/8thj | HTTPS FTP |
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-Related structure data
Related structure data | 41266MC 8thiC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 / #3: Chemical | #4: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: HiSiaQM transporter protein solubilised in amphipol A8-35 Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Haemophilus influenzae Rd KW20 (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: TOP10 |
Buffer solution | pH: 8 |
Specimen | Conc.: 4.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm |
Image recording | Electron dose: 73.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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Symmetry | Point symmetry: C2 (2 fold cyclic) |
3D reconstruction | Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225044 / Symmetry type: POINT |