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- EMDB-33756: Structure of PfNT1(Y190A)-GFP in complex with GSK4 -

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Basic information

Entry
Database: EMDB / ID: EMD-33756
TitleStructure of PfNT1(Y190A)-GFP in complex with GSK4
Map data
Sample
  • Complex: nucleoside/nucleobase transporter fusion with GFP
    • Protein or peptide: Nucleoside transporter 1,Green fluorescent protein
  • Ligand: 5-methyl-N-[2-(2-oxidanylideneazepan-1-yl)ethyl]-2-phenyl-1,3-oxazole-4-carboxamide
Keywordsmalaria / nucleoside transporter / GSK4 / TRANSPORT PROTEIN / MEMBRANE PROTEIN
Function / homology
Function and homology information


Transport of nucleosides and free purine and pyrimidine bases across the plasma membrane / Ribavirin ADME / adenosine transport / Azathioprine ADME / nucleoside transmembrane transporter activity / purine nucleobase transport / bioluminescence / generation of precursor metabolites and energy / plasma membrane
Similarity search - Function
Equilibrative nucleoside transporter / MFS transporter superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Green fluorescent protein / Nucleoside transporter 1
Similarity search - Component
Biological speciesPlasmodium falciparum 3D7 (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.04 Å
AuthorsWang C / Yu LY / Li JL / Ren RB / Deng D
Funding support China, 2 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)National Key R&D Program of China 2016YFA0502700 China
National Natural Science Foundation of China (NSFC)32171211 China
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1.
Authors: Chen Wang / Leiye Yu / Jiying Zhang / Yanxia Zhou / Bo Sun / Qingjie Xiao / Minhua Zhang / Huayi Liu / Jinhong Li / Jialu Li / Yunzi Luo / Jie Xu / Zhong Lian / Jingwen Lin / Xiang Wang / ...Authors: Chen Wang / Leiye Yu / Jiying Zhang / Yanxia Zhou / Bo Sun / Qingjie Xiao / Minhua Zhang / Huayi Liu / Jinhong Li / Jialu Li / Yunzi Luo / Jie Xu / Zhong Lian / Jingwen Lin / Xiang Wang / Peng Zhang / Li Guo / Ruobing Ren / Dong Deng /
Abstract: By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates ...By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.
History
DepositionJul 4, 2022-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33756.png

Downloads & links

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Map

FileDownload / File: emd_33756.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-1.5396911 - 2.1936698
Average (Standard dev.)0.0026815631 (±0.04239917)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 215.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_33756_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33756_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nucleoside/nucleobase transporter fusion with GFP

EntireName: nucleoside/nucleobase transporter fusion with GFP
Components
  • Complex: nucleoside/nucleobase transporter fusion with GFP
    • Protein or peptide: Nucleoside transporter 1,Green fluorescent protein
  • Ligand: 5-methyl-N-[2-(2-oxidanylideneazepan-1-yl)ethyl]-2-phenyl-1,3-oxazole-4-carboxamide

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Supramolecule #1: nucleoside/nucleobase transporter fusion with GFP

SupramoleculeName: nucleoside/nucleobase transporter fusion with GFP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: the cDNA of the GFP was cloned into PfNT1 between K370 and K371
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)
Molecular weightTheoretical: 47.5 kDa/nm

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Macromolecule #1: Nucleoside transporter 1,Green fluorescent protein

MacromoleculeName: Nucleoside transporter 1,Green fluorescent protein / type: protein_or_peptide / ID: 1
Details: PfNT1(Y190A) fused with GFP the GFP was inserted into PfNT1 between K370 and K371
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)
Molecular weightTheoretical: 77.035766 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHHHH HSGDEVDAGS GHMSTGKESS KAYADIESRG DYKDDGKKGS TLSSKQHFML SLTFILIGLS SLNVWNTALG LNINFKYNT FQITGLVCSS IVALFVEIPK IMLPFLLGGL SILCAGFQIS HSFFTDTQFD TYCLVAFIVI GVVAGLAQTI A FNIGSTME ...String:
MHHHHHHHHH HSGDEVDAGS GHMSTGKESS KAYADIESRG DYKDDGKKGS TLSSKQHFML SLTFILIGLS SLNVWNTALG LNINFKYNT FQITGLVCSS IVALFVEIPK IMLPFLLGGL SILCAGFQIS HSFFTDTQFD TYCLVAFIVI GVVAGLAQTI A FNIGSTME DNMGGYMSAG IGISGVFIFV INLLLDQFVS PEKHYGVNKA KLLALYIICE LCLILAIVFC VCNLDLTNKN NK KDEENKE NNATLSYMEL FKDSYKAILT MFLVNWLTLQ LFPGVGHKKW QESHNISDYN VTIIVGMFQV FDFLSRYPPN LTH IKIFKN FTFSLNKLLV ANSLRLLFIP WFILNACVDH PFFKNIVQQC VCMAMLAFTN GWFNTVPFLV FVKELKMVSK GEEL FTGVV PILVELDGDV NGHKFSVSGE GEGDATYGKL TLKFICTTGK LPVPWPTLVT TLTYGVQCFS RYPDHMKQHD FFKSA MPEG YVQERTIFFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYNSHNVY IMADKQKNGI KANFKI RHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKKA KKKKEIE II STFLVIAMFV GLFCGIWTTY IYNLFNIVLP KPDLPPIDVT Q

UniProtKB: Nucleoside transporter 1, Green fluorescent protein, Nucleoside transporter 1

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Macromolecule #2: 5-methyl-N-[2-(2-oxidanylideneazepan-1-yl)ethyl]-2-phenyl-1,3-oxa...

MacromoleculeName: 5-methyl-N-[2-(2-oxidanylideneazepan-1-yl)ethyl]-2-phenyl-1,3-oxazole-4-carboxamide
type: ligand / ID: 2 / Number of copies: 1 / Formula: IRX
Molecular weightTheoretical: 341.404 Da
Chemical component information

ChemComp-IRX:
5-methyl-N-[2-(2-oxidanylideneazepan-1-yl)ethyl]-2-phenyl-1,3-oxazole-4-carboxamide

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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

BufferpH: 6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 52.452 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: 1.1 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 329768
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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