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- PDB-7zc2: Dipeptide and tripeptide Permease C (DtpC) -

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Basic information

Entry
Database: PDB / ID: 7zc2
TitleDipeptide and tripeptide Permease C (DtpC)
ComponentsAmino acid/peptide transporter
KeywordsTRANSPORT PROTEIN / Membrane protein / Peptide transporter / Proton coupled oligopeptide transporter / POT / MFS / DtpC.
Function / homology
Function and homology information


oligopeptide transport / peptide transmembrane transporter activity / membrane => GO:0016020 / 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
Amino acid/peptide transporter
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsKiller, M. / Finocchio, G. / Pardon, E. / Steyaert, J. / Loew, C.
Funding support1items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Amino acid/peptide transporter


Theoretical massNumber of molelcules
Total (without water)53,0901
Polymers53,0901
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Dipeptide and tripeptide permease C (DtpC) also forms dimers and each copy binds to the conformation specific nanobody 26.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area22660 Å2

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Components

#1: Protein Amino acid/peptide transporter / Di/tripeptide permease YjdL / Dipeptide and tripeptide permease / Dipeptide and tripeptide permease ...Di/tripeptide permease YjdL / Dipeptide and tripeptide permease / Dipeptide and tripeptide permease D / Dipeptide/tripeptide permease DtpC / MFS transporter / Peptide permease / Probable dipeptide and tripeptide permease YjdL / Putative dipeptide and tripeptide permease YjdL / YjdL peptide transporter


Mass: 53090.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: dtpD_2, dtpC, dtpD_1, yjdL, AM464_14395, AT845_002896, AWP93_17895, BANRA_02817, BGZ_01664, BGZ_05066, BJI68_16865, BJJ90_24000, BO068_004094, BON64_05130, BON69_04615, BON71_15930, BON73_ ...Gene: dtpD_2, dtpC, dtpD_1, yjdL, AM464_14395, AT845_002896, AWP93_17895, BANRA_02817, BGZ_01664, BGZ_05066, BJI68_16865, BJJ90_24000, BO068_004094, BON64_05130, BON69_04615, BON71_15930, BON73_07300, BON74_00740, BON75_25330, BON77_18065, BON80_13570, BON89_26025, BON93_12630, BON97_19640, BvCmsF30A_02788, BvCmsHHP019_00793, BvCmsHHP056_00023, BvCmsKKP061_00271, C2U48_11920, C3F40_14675, C5N07_06940, C5Y87_09615, CA593_05175, CG831_003409, CIG67_08950, COD53_12960, CWS33_03315, D0X26_12710, D9D77_08965, DAH17_08915, DAH18_16490, DAH34_18055, DAH35_18055, DAH36_09805, DAH37_06440, DEN87_12425, DEO12_13910, DEO20_13520, DXT71_14295, DXT73_07155, E0I42_01290, E2119_15085, E2121_02870, E2122_03065, E2131_09650, E2135_09870, E4K51_08850, E5M02_11420, E5P26_01815, E5P27_12215, E5P28_03135, E5P29_19355, E5P31_11330, E5P32_03320, E5P33_11435, E5P35_07320, E5P36_20530, E5P40_16720, E5S34_00615, E5S35_05230, E5S36_08360, E5S37_06350, E5S42_15010, E5S43_14690, E5S45_06265, E5S51_06500, E5S54_12820, E5S57_19065, EC1094V2_4123, EC3234A_78c00250, EI021_09455, EIZ93_10840, EL79_4257, EL80_4164, ELT20_09370, ELT41_02450, ELV08_11600, ELY39_09460, ERS139208_00804, EYV17_05270, EYV18_19620, F2N31_14570, F9V24_13955, F9X20_06665, FDM60_18295, FOI11_014975, FOI11_20705, FQ007_15450, FQF29_19850, FV293_00630, G5632_13435, GF699_20045, GIB53_13155, GKF89_08690, GKG12_11445, GNZ05_19605, GP662_06300, GQF59_12650, GRW05_17855, GRW57_10485, GRW81_08175, H0O72_04690, HHH44_003360, HMV95_08810, HNC36_09415, HV109_21715, HVY77_23340, HX136_22920, I6H02_16470, IH768_20930, IH772_00050, J0541_004579, J4S20_000461, JFD_00784, JNP96_02330, NCTC10429_06294, NCTC11126_02220, NCTC11181_01802, NCTC13216_02261, NCTC8008_04125, NCTC9037_04624, NCTC9045_05367, NCTC9073_04059, PGD_03312, SAMEA3472067_03850, SAMEA3472080_00141, SAMEA3752557_02097, WP2S18E08_44130, WR15_07240
Production host: Escherichia coli (E. coli) / References: UniProt: W8TWZ0

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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: Dipeptide and tripeptide permease C (DtpC) / Type: CELL / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 75 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 24333
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6464070
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 878428 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 12.73 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00283719
ELECTRON MICROSCOPYf_angle_d0.61625057
ELECTRON MICROSCOPYf_chiral_restr0.0391586
ELECTRON MICROSCOPYf_plane_restr0.0079621
ELECTRON MICROSCOPYf_dihedral_angle_d17.08251265

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