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- EMDB-9832: Tetrameric PepTSo2 incorporated in salipro nano particle -

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Basic information

Entry
Database: EMDB / ID: EMD-9832
TitleTetrameric PepTSo2 incorporated in salipro nano particle
Map data
Sample
  • Complex: Tetrameric PepTSo2 with saposin A
    • Protein or peptide: Proton:oligopeptide symporter POT family
KeywordsPeptide transporter / MEMBRANE PROTEIN
Function / homology
Function and homology information


dipeptide transmembrane transport / tripeptide transmembrane transporter activity / peptide:proton symporter activity / dipeptide transmembrane transporter activity / membrane / identical protein binding
Similarity search - Function
Dipeptide/tripeptide permease / Proton-dependent oligopeptide transporter family / POT family / MFS transporter superfamily
Similarity search - Domain/homology
Proton:oligopeptide symporter POT family
Similarity search - Component
Biological speciesShewanella oneidensis (bacteria) / Shewanella oneidensis MR-1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKawamoto A / Matoba K
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science24227004 Japan
Japan Society for the Promotion of Science25291011 Japan
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.
History
DepositionFeb 19, 2019-
Header (metadata) releaseMay 15, 2019-
Map releaseMay 15, 2019-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ji1
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9832.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.09303759 - 0.1250117
Average (Standard dev.)0.00031285142 (±0.0036033548)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 264.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z304304304
origin x/y/z0.0000.0000.000
length x/y/z264.480264.480264.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS304304304
D min/max/mean-0.0930.1250.000

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Supplemental data

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Sample components

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Entire : Tetrameric PepTSo2 with saposin A

EntireName: Tetrameric PepTSo2 with saposin A
Components
  • Complex: Tetrameric PepTSo2 with saposin A
    • Protein or peptide: Proton:oligopeptide symporter POT family

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Supramolecule #1: Tetrameric PepTSo2 with saposin A

SupramoleculeName: Tetrameric PepTSo2 with saposin A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Shewanella oneidensis (bacteria)

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Macromolecule #1: Proton:oligopeptide symporter POT family

MacromoleculeName: Proton:oligopeptide symporter POT family / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Shewanella oneidensis MR-1 (bacteria) / Strain: MR-1
Molecular weightTheoretical: 57.626559 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MTLGTNQVSK THSFMTVSLI ELWERFGYYG MQALIVYFMV QRLGFDDSRA NLVWSACAAL IYVSPAIGGW VGDKILGTKR TMLLGAGIL SVGYALMTVP TENTWFMFSA LGVIVVGNGL FKPNAGNLVR KIYEGDDSKI DSAFTIYYMA VNVGSTFSML L TPWIKDYV ...String:
MTLGTNQVSK THSFMTVSLI ELWERFGYYG MQALIVYFMV QRLGFDDSRA NLVWSACAAL IYVSPAIGGW VGDKILGTKR TMLLGAGIL SVGYALMTVP TENTWFMFSA LGVIVVGNGL FKPNAGNLVR KIYEGDDSKI DSAFTIYYMA VNVGSTFSML L TPWIKDYV NAQYGNEFGW HAAFAVCCVG ILVGLGNYAL MHKSLANYGS EPDTRPVNKK SLAIVLALAA LSVVASAIIL EY EDVARVF VYAAGVAVLG IFFHLIRTSE PSERAGLIAA LILTVQTVFF FIFYQQMSTS LALFALRNVD WDFQVFGTHL WTW SPAQFQ ALNPIWIMVL SPVLAWSYSW AGRNNKDFSI AAKFALGFAV VAIGFFIYGF AGQFAVNGKT SSWVMIWGYA SYSL GELLV SGLGLAMIAR YVPARMGGFM MGAYFVASGI SQYLGGVVAN FASVPQDLVD PLQTLPVYTN LFNKLGVAAV VCTII ALAV LPLMRRLTES HHAHSSIENN AAASLRDVKA EQLESSGENL YFQ

UniProtKB: Proton:oligopeptide symporter POT family

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMNa-phosphatesodium phosphate
200.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blotted for 4.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 96000
Sample stageCooling holder cryogen: NITROGEN
TemperatureMin: 79.55 K / Max: 79.55 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 1609 / Average electron dose: 82.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 288417
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
Final 3D classificationSoftware - Name: RELION (ver. 3.0-beta)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0-beta)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta) / Number images used: 43172
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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