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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
SampleTetrameric PepTSo2 with saposin A
  • Proton:oligopeptide symporter POT family
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
SourceShewanella oneidensis (bacteria) / Shewanella oneidensis MR-1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKawamoto A / Matoba K / Takagi J
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 ReportPDB-ID: 6ji1

SummaryFull reportAbout validation report
DateDeposition: Feb 19, 2019 / Header (metadata) release: May 15, 2019 / Map release: May 15, 2019 / Update: May 15, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • 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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 304 pix.
= 264.48 Å
0.87 Å/pix.
x 304 pix.
= 264.48 Å
0.87 Å/pix.
x 304 pix.
= 264.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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 / Number of components: 2

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Component #1: protein, Tetrameric PepTSo2 with saposin A

ProteinName: Tetrameric PepTSo2 with saposin A / Recombinant expression: No
SourceSpecies: Shewanella oneidensis (bacteria)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria) / Strain: K-12

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Component #2: protein, Proton:oligopeptide symporter POT family

ProteinName: Proton:oligopeptide symporter POT family / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 57.626559 kDa
SourceSpecies: Shewanella oneidensis MR-1 (bacteria) / Strain: MR-1
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 % / Details: blotted for 4.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 82 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 96000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 3000.0 nm
Specimen HolderModel: OTHER / Temperature: (79.55 - 79.55 K)
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1609

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C4 (4 fold cyclic) / Number of projections: 43172
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Output model

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