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- PDB-6ji1: Tetrameric PepTSo2 incorporated in salipro nano particle -

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Entry
Database: PDB / ID: 6ji1
TitleTetrameric PepTSo2 incorporated in salipro nano particle
ComponentsProton:oligopeptide symporter POT family
KeywordsMEMBRANE PROTEIN / Peptide transporter
Function / homology
Function and homology information


tripeptide transmembrane transporter activity / peptide:proton symporter activity / oligopeptide transmembrane transporter activity / dipeptide transmembrane transporter activity / peptide transmembrane transporter activity / integral component of plasma membrane / integral component of membrane / identical protein binding / plasma membrane
Dipeptide/tripeptide permease / Proton-dependent oligopeptide transporter family / Major facilitator superfamily domain / MFS transporter superfamily / POT family
Proton:oligopeptide symporter POT family
Specimen sourceShewanella oneidensis MR-1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKawamoto, A. / Matoba, K. / Takagi, J.
Funding supportJapan , 2件
OrganizationGrant numberCountry
Japan Society for the Promotion of Science24227004Japan
Japan Society for the Promotion of Science25291011Japan
CitationJournal: Acta Crystallogr F Struct Biol Commun / Year: 2019
Title: Structural basis for oligomerization of the prokaryotic peptide transporter PepT.
Authors: Reina Nagamura / Masahiro Fukuda / Akihiro Kawamoto / Kyoko Matoba / Naoshi Dohmae / Ryuichiro Ishitani / Junichi Takagi / Osamu Nureki /
Abstract: Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. ...Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1-H6 and H7-H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepT from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepT-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepT were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepT incorporated into Salipro nanoparticles at 4.1 Å resolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepT to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepT, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 19, 2019 / Release: May 15, 2019Array

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Assembly

Deposited unit
A: Proton:oligopeptide symporter POT family
B: Proton:oligopeptide symporter POT family
C: Proton:oligopeptide symporter POT family
D: Proton:oligopeptide symporter POT family


Theoretical massNumber of molelcules
Total (without water)230,5064
Polymers230,5064
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6000 Å2
ΔGint-53 kcal/mol
Surface area85910 Å2

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Components

#1: Protein/peptide
Proton:oligopeptide symporter POT family


Mass: 57626.559 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shewanella oneidensis MR-1 (bacteria) / Strain: MR-1 / Gene: SO_1277 / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): K-12 / References: UniProt: Q8EHE6

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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: Tetrameric PepTSo2 with saposin A / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Shewanella oneidensis (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Buffer solutionpH: 7.4
Buffer component

Buffer-ID: 1

IDConc.NameFormula
125 mMsodium phosphateNa-phosphate
2200 mMsodium chlorideNaCl
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blotted for 4.5 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Temperature (max): 79.55 K / Temperature (min): 79.55 K
Image recordingElectron dose: 82 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 1609
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: (1.13_2998: phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9RELION3.0-betainitial Euler assignment
10RELION3.0-betafinal Euler assignment
11RELION3.0-betaclassification
12RELION3.0-beta3D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 288417
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43172 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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