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- PDB-7qha: Cryo-EM structure of the Tripartite ATP-independent Periplasmic (... -

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Basic information

Entry
Database: PDB / ID: 7qha
TitleCryo-EM structure of the Tripartite ATP-independent Periplasmic (TRAP) transporter SiaQM from Photobacterium profundum in amphipol
Components
  • Megabody c7HopQ
  • Putative TRAP-type C4-dicarboxylate transport system, large permease component
  • Putative TRAP-type C4-dicarboxylate transport system, small permease component
KeywordsTRANSPORT PROTEIN / TRAP / elevator / secondary transporter / sialic acid
Function / homology
Function and homology information


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 / SabA, N-terminal extracellular adhesion domain / SabA N-terminal extracellular adhesion domain / Outer membrane protein, Helicobacter / Helicobacter outer membrane protein
Similarity search - Domain/homology
Outer membrane protein / TRAP transporter small permease protein / TRAP transporter large permease protein
Similarity search - Component
Biological speciesPhotobacterium profundum SS9 (bacteria)
Helicobacter pylori (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsNorth, R.A. / Davies, J.S. / Morado, D. / Dobson, R.C.J.
Funding support New Zealand, 2items
OrganizationGrant numberCountry
Marsden FundUOC1506 New Zealand
Ministry of Business, Innovation and Employment (New Zealand)UOCX1706 New Zealand
CitationJournal: Nat Commun / Year: 2023
Title: Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.
Authors: James S Davies / Michael J Currie / Rachel A North / Mariafrancesca Scalise / Joshua D Wright / Jack M Copping / Daniela M Remus / Ashutosh Gulati / Dustin R Morado / Sam A Jamieson / ...Authors: James S Davies / Michael J Currie / Rachel A North / Mariafrancesca Scalise / Joshua D Wright / Jack M Copping / Daniela M Remus / Ashutosh Gulati / Dustin R Morado / Sam A Jamieson / Michael C Newton-Vesty / Gayan S Abeysekera / Subramanian Ramaswamy / Rosmarie Friemann / Soichi Wakatsuki / Jane R Allison / Cesare Indiveri / David Drew / Peter D Mace / Renwick C J Dobson /
Abstract: In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding ...In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.
History
DepositionDec 11, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Putative TRAP-type C4-dicarboxylate transport system, small permease component
B: Putative TRAP-type C4-dicarboxylate transport system, large permease component
C: Megabody c7HopQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,2355
Polymers120,1893
Non-polymers462
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7290 Å2
ΔGint-71 kcal/mol
Surface area30370 Å2
MethodPISA

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Components

#1: Protein Putative TRAP-type C4-dicarboxylate transport system, small permease component


Mass: 19748.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photobacterium profundum SS9 (bacteria)
Strain: SS9 / Gene: VC1778, PBPRA2280 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6LPW0
#2: Protein Putative TRAP-type C4-dicarboxylate transport system, large permease component


Mass: 45573.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photobacterium profundum SS9 (bacteria)
Strain: SS9 / Gene: SMB20297, PBPRA2279 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6LPW1
#3: Antibody Megabody c7HopQ


Mass: 54867.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicobacter pylori (bacteria), (gene. exp.) synthetic construct (others)
Strain: G27 / Gene: hopQ, HPG27_1120 / Production host: Escherichia coli (E. coli) / References: UniProt: B5Z8H1
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1SiaQM with megabodyCOMPLEX#1-#30MULTIPLE SOURCES
2SiaQMCOMPLEX#1-#21RECOMBINANT
3MegabodyCOMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Photobacterium profundum (bacteria)74109
33Helicobacter pylori (bacteria)210
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenConc.: 2.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm
Image recordingAverage exposure time: 1.9 sec. / Electron dose: 71.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10960

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 624033 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035405
ELECTRON MICROSCOPYf_angle_d0.5737352
ELECTRON MICROSCOPYf_dihedral_angle_d3.785732
ELECTRON MICROSCOPYf_chiral_restr0.041880
ELECTRON MICROSCOPYf_plane_restr0.004897

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