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- EMDB-14618: Dipeptide and tripeptide Permease C (DtpC) -

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Basic information

Entry
Database: EMDB / ID: EMD-14618
TitleDipeptide and tripeptide Permease C (DtpC)
Map dataPost-processed map. Focused ref on one copy of DtpC.
Sample
  • Cell: Dipeptide and tripeptide permease C (DtpC)
    • Protein or peptide: Amino acid/peptide transporter
KeywordsMembrane protein / Peptide transporter / Proton coupled oligopeptide transporter / POT / MFS / DtpC. / TRANSPORT PROTEIN
Function / homology
Function and homology information


oligopeptide transport / peptide transmembrane transporter activity / peptide transport / plasma membrane
Similarity search - Function
: / Dipeptide/tripeptide permease / PTR2 family proton/oligopeptide symporters signature 1. / PTR2 family proton/oligopeptide symporters signature 2. / PTR2 family proton/oligopeptide symporter, conserved site / Proton-dependent oligopeptide transporter family / POT family / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsKiller M / Finocchio G / Pardon E / Steyaert J / Loew C
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Front Mol Biosci / Year: 2022
Title: Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C.
Authors: Maxime Killer / Giada Finocchio / Haydyn D T Mertens / Dmitri I Svergun / Els Pardon / Jan Steyaert / Christian Löw /
Abstract: Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the ...Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa Pro-macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 Å. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.
History
DepositionMar 25, 2022-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14618.png

Downloads & links

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Map

FileDownload / File: emd_14618.map.gz / Format: CCP4 / Size: 5.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map. Focused ref on one copy of DtpC.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.85 Å/pix.
x 115 pix.
= 97.75 Å		0.85 Å/pix.
x 115 pix.
= 97.75 Å		0.85 Å/pix.
x 113 pix.
= 96.05 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum-9.466241999999999 - 18.653155999999999
Average (Standard dev.)-0.000000000002885 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin147161141
Dimensions115113115
Spacing115115113
CellA: 97.75 Å / B: 97.75 Å / C: 96.05 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14618_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post-processed map. Dimeric reconstruction in complex with nanobody...

Fileemd_14618_additional_1.map
AnnotationPost-processed map. Dimeric reconstruction in complex with nanobody 26.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map 1. Dimeric reconstruction in complex with nanobody 26.

Fileemd_14618_additional_2.map
AnnotationHalf map 1. Dimeric reconstruction in complex with nanobody 26.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map 2. Dimeric reconstruction in complex with nanobody 26.

Fileemd_14618_additional_3.map
AnnotationHalf map 2. Dimeric reconstruction in complex with nanobody 26.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1. Focused ref on one copy of DtpC.

Fileemd_14618_half_map_1.map
AnnotationHalf map 1. Focused ref on one copy of DtpC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2. Focused ref on one copy of DtpC.

Fileemd_14618_half_map_2.map
AnnotationHalf map 2. Focused ref on one copy of DtpC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dipeptide and tripeptide permease C (DtpC)

EntireName: Dipeptide and tripeptide permease C (DtpC)
Components
  • Cell: Dipeptide and tripeptide permease C (DtpC)
    • Protein or peptide: Amino acid/peptide transporter

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Supramolecule #1: Dipeptide and tripeptide permease C (DtpC)

SupramoleculeName: Dipeptide and tripeptide permease C (DtpC) / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Amino acid/peptide transporter

MacromoleculeName: Amino acid/peptide transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 53.090273 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKTPSQPRAI YYIVAIQIWE YFSFYGMRAL LILYLTHQLG FDDNHAISLF SAYASLVYVT PILGGWLADR LLGNRTAVIA GALLMTLGH VVLGIDTNST FSLYLALAII ICGYGLFKSN ISCLLGELYD ENDHRRDGGF SLLYAAGNIG SIAAPIACGL A AQWYGWHV ...String:
MKTPSQPRAI YYIVAIQIWE YFSFYGMRAL LILYLTHQLG FDDNHAISLF SAYASLVYVT PILGGWLADR LLGNRTAVIA GALLMTLGH VVLGIDTNST FSLYLALAII ICGYGLFKSN ISCLLGELYD ENDHRRDGGF SLLYAAGNIG SIAAPIACGL A AQWYGWHV GFALAGGGMF IGLLIFLSGH RHFQSTRSMD KKALTSVKFA LPVWSWLVVM LCLAPVFFTL LLENDWSGYL LA IVCLIAA QIIARMMIKF PEHRRALWQI VLLMFVGTLF WVLAQQGGST ISLFIDRFVN RQAFNIEVPT ALFQSVNAIA VML AGVVLA WLASPESRGN STLRVWLKFA FGLLLMACGF MLLAFDARHA AADGQASMGV MISGLALMGF AELFIDPVAI AQIT RLKMS GVLTGIYMLA TGAVANWLAG VVAQQTTESQ ISGMAIAAYQ RFFSQMGEWT LACVAIIVVL AFATRFLFST PTNMI QESN D

UniProtKB: Peptide permease

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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

Concentration6 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 24333 / Average electron dose: 75.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6464070
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 878428
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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