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- EMDB-32619: Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine -

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Basic information

Entry
Database: EMDB / ID: EMD-32619
TitleStructure of PfNT1(Y190A) in complex with nanobody 48 and inosine
Map data
Sample
  • Complex: Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine
    • Complex: PfNT1
      • Protein or peptide: Equilibrative nucleoside/nucleobase transporter
    • Complex: nanobody 48
      • Protein or peptide: nanobody48
  • Ligand: INOSINE
Keywordsmalaria / Equilibrative nucleoside transporter / inosine / TRANSPORT PROTEIN
Function / homologynicotinamide riboside transmembrane transporter activity / Equilibrative nucleoside transporter / nucleobase transmembrane transporter activity / MFS transporter superfamily / membrane / Equilibrative nucleoside/nucleobase transporter
Function and homology information
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsWang C / Deng D
Funding support China, 2 items
OrganizationGrant numberCountry
National Key R&D Program of China2016YFA0502700 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
DepositionJan 17, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32619.png

Downloads & links

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Map

FileDownload / File: emd_32619.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.313
Minimum - Maximum-0.64293295 - 1.3155411
Average (Standard dev.)-0.0001925501 (±0.032868512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine

EntireName: Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine
Components
  • Complex: Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine
    • Complex: PfNT1
      • Protein or peptide: Equilibrative nucleoside/nucleobase transporter
    • Complex: nanobody 48
      • Protein or peptide: nanobody48
  • Ligand: INOSINE

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Supramolecule #1: Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine

SupramoleculeName: Structure of PfNT1(Y190A) in complex with nanobody 48 and inosine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Plasmodium falciparum (malaria parasite P. falciparum)

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Supramolecule #2: PfNT1

SupramoleculeName: PfNT1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Vicugna pacos (alpaca)

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Supramolecule #3: nanobody 48

SupramoleculeName: nanobody 48 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Equilibrative nucleoside/nucleobase transporter

MacromoleculeName: Equilibrative nucleoside/nucleobase transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Plasmodium falciparum (malaria parasite P. falciparum)
Molecular weightTheoretical: 47.579801 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSTGKESSKA YADIESRGDY KDDGKKGSTL SSKQHFMLSL TFILIGLSSL NVWNTALGLN INFKYNTFQI TGLVCSSIVA LFVEIPKIM LPFLLGGLSI LCAGFQISHS FFTDTQFDTY CLVAFIVIGV VAGLAQTIAF NIGSTMEDNM GGYMSAGIGI S GVFIFVIN ...String:
MSTGKESSKA YADIESRGDY KDDGKKGSTL SSKQHFMLSL TFILIGLSSL NVWNTALGLN INFKYNTFQI TGLVCSSIVA LFVEIPKIM LPFLLGGLSI LCAGFQISHS FFTDTQFDTY CLVAFIVIGV VAGLAQTIAF NIGSTMEDNM GGYMSAGIGI S GVFIFVIN LLLDQFVSPE KHYGVNKAKL LALYIICELC LILAIVFCVC NLDLTNKNNK KDEENKENNA TLSYMELFKD SY KAILTMF LVNWLTLQLF PGVGHKKWQE SHNISDYNVT IIVGMFQVFD FLSRYPPNLT HIKIFKNFTF SLNKLLVANS LRL LFIPWF ILNACVDHPF FKNIVQQCVC MAMLAFTNGW FNTVPFLVFV KELKKAKKKK EIEIISTFLV IAMFVGLFCG IWTT YIYNL FNIVLPKPDL PPIDVTQ

UniProtKB: Equilibrative nucleoside/nucleobase transporter

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Macromolecule #2: nanobody48

MacromoleculeName: nanobody48 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.199559 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString:
QVQLVESGGG LVQPGGSLRL SCAASGFTGS INYMGWYRQA PGKQRELVAR FSSGGSTNYA DSVKGRFTIS GDNAKNTVYL QMNSLKPED TAVYYCNAET ISYVYTVVFQ DYWGQGTQVT VSS

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Macromolecule #3: INOSINE

MacromoleculeName: INOSINE / type: ligand / ID: 3 / Number of copies: 1 / Formula: NOS
Molecular weightTheoretical: 268.226 Da
Chemical component information

ChemComp-NOS:
INOSINE

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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 K3 (6k x 4k) / Average electron dose: 56.3 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: 1.0 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: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 183797
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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