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- PDB-7t3e: Structure of the sialic acid bound Tripartite ATP-independent Per... -

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Basic information

Entry
Database: PDB / ID: 7t3e
TitleStructure of the sialic acid bound Tripartite ATP-independent Periplasmic (TRAP) periplasmic component SiaP from Photobacterium profundum
ComponentsTRAP-type C4-dicarboxylate transport system, periplasmic component
KeywordsTRANSPORT PROTEIN / Periplasmic SBP / Sialic acid / TRAP transporter
Function / homology
Function and homology information


transmembrane transport / outer membrane-bounded periplasmic space
Similarity search - Function
TRAP transporter solute receptor, DctP family / Bacterial extracellular solute-binding protein, family 7 / TRAP transporter solute receptor DctP / TRAP transporter solute receptor DctP superfamily / Bacterial extracellular solute-binding protein, family 7 / D-Maltodextrin-Binding Protein; domain 2 / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
N-acetyl-beta-neuraminic acid / TRAP-type C4-dicarboxylate transport system, periplasmic component
Similarity search - Component
Biological speciesPhotobacterium profundum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.04 Å
AuthorsDavies, J.S. / Currie, M.J. / North, R.A. / 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 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: Database references / Structure summary / Category: citation / citation_author / struct
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 / _struct.title
Revision 1.2Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TRAP-type C4-dicarboxylate transport system, periplasmic component
B: TRAP-type C4-dicarboxylate transport system, periplasmic component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,6746
Polymers67,8632
Non-polymers8114
Water14,394799
1
A: TRAP-type C4-dicarboxylate transport system, periplasmic component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,3373
Polymers33,9321
Non-polymers4052
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: TRAP-type C4-dicarboxylate transport system, periplasmic component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,3373
Polymers33,9321
Non-polymers4052
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)58.596, 87.313, 129.412
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein TRAP-type C4-dicarboxylate transport system, periplasmic component / SiaP


Mass: 33931.586 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photobacterium profundum (bacteria) / Gene: SMB20295, PBPRA2281 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6LPV9
#2: Sugar ChemComp-SLB / N-acetyl-beta-neuraminic acid / N-acetylneuraminic acid / sialic acid / O-sialic acid / 5-N-ACETYL-BETA-D-NEURAMINIC ACID / BETA-SIALIC ACID


Type: D-saccharide, beta linking / Mass: 309.270 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H19NO9 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DNeup5AcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-neuraminic acidCOMMON NAMEGMML 1.0
b-D-Neup5AcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
Neu5AcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 799 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.44 Å3/Da / Density % sol: 49.57 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop
Details: 50 mg/mL protein in 50 mM Tris, pH 8.0, 150 mM sodium chloride, 0.002% w/v L-MNG mixed 1:1 with 0.1 M Bis-Tris, pH 5.0, 2 M ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95372 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 9, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95372 Å / Relative weight: 1
ReflectionResolution: 1.04→43.66 Å / Num. obs: 316707 / % possible obs: 99.6 % / Redundancy: 6.5 % / Biso Wilson estimate: 10.24 Å2 / Rmerge(I) obs: 0.073 / Net I/σ(I): 9.8
Reflection shellResolution: 1.04→1.077 Å / Redundancy: 5.7 % / Rmerge(I) obs: 1.153 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 31310 / % possible all: 93

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
Cootmodel building
PDB-REDOrefinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2xwk

2xwk


Resolution: 1.04→43.66 Å / SU ML: 0.0931 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 13.4518
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1386 30762 4.99 %
Rwork0.1237 585192 -
obs0.1245 316707 99.36 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 14.49 Å2
Refinement stepCycle: LAST / Resolution: 1.04→43.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4768 0 52 799 5619
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0095601
X-RAY DIFFRACTIONf_angle_d1.1127692
X-RAY DIFFRACTIONf_chiral_restr0.0855836
X-RAY DIFFRACTIONf_plane_restr0.01051039
X-RAY DIFFRACTIONf_dihedral_angle_d5.2598794
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.04-1.050.29538780.309117086X-RAY DIFFRACTION86.71
1.05-1.060.294610420.278419625X-RAY DIFFRACTION99.86
1.06-1.080.263310120.257919550X-RAY DIFFRACTION99.83
1.08-1.090.24649630.241419635X-RAY DIFFRACTION99.81
1.09-1.10.223510080.222519641X-RAY DIFFRACTION99.8
1.1-1.120.2139770.204919643X-RAY DIFFRACTION99.69
1.12-1.130.19829540.195319496X-RAY DIFFRACTION99.15
1.13-1.150.18219930.181519528X-RAY DIFFRACTION99.34
1.15-1.170.188310360.170519724X-RAY DIFFRACTION99.97
1.17-1.190.181211440.167419414X-RAY DIFFRACTION99.9
1.19-1.210.170910250.154119648X-RAY DIFFRACTION99.97
1.21-1.230.163110000.147919585X-RAY DIFFRACTION99.95
1.23-1.250.152710130.141519661X-RAY DIFFRACTION99.93
1.25-1.280.140211130.1319576X-RAY DIFFRACTION99.95
1.28-1.310.154410600.123119537X-RAY DIFFRACTION99.87
1.31-1.340.139410020.113519620X-RAY DIFFRACTION99.86
1.34-1.370.123210510.106219567X-RAY DIFFRACTION99.78
1.37-1.410.12410240.102619608X-RAY DIFFRACTION99.71
1.41-1.450.12129840.098719443X-RAY DIFFRACTION98.74
1.45-1.50.11810440.095919578X-RAY DIFFRACTION99.71
1.5-1.550.115910170.093819576X-RAY DIFFRACTION99.91
1.55-1.610.100310880.082919560X-RAY DIFFRACTION99.95
1.61-1.690.110311030.081519596X-RAY DIFFRACTION99.99
1.69-1.770.111610350.08619691X-RAY DIFFRACTION100
1.77-1.890.10089760.092319619X-RAY DIFFRACTION100
1.89-2.030.12139960.098819691X-RAY DIFFRACTION100
2.03-2.240.113610090.099319664X-RAY DIFFRACTION100
2.24-2.560.112410640.107519512X-RAY DIFFRACTION99.52
2.56-3.220.1410600.128419554X-RAY DIFFRACTION99.91
3.23-43.660.149210910.134519564X-RAY DIFFRACTION99.96

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