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- PDB-8tz6: Cryo-EM structure of bovine concentrative nucleoside transporter ... -

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Basic information

Entry
Database: PDB / ID: 8tz6
TitleCryo-EM structure of bovine concentrative nucleoside transporter 3 in complex with PSI-6206
ComponentsSodium/nucleoside cotransporter
KeywordsTRANSPORT PROTEIN / membrane protein / transporter / nucleoside
Function / homology
Function and homology information


Ribavirin ADME / purine-specific nucleoside:sodium symporter activity / Transport of nucleosides and free purine and pyrimidine bases across the plasma membrane / pyrimidine nucleoside transport / pyrimidine- and adenosine-specific:sodium symporter activity / purine nucleoside transmembrane transport / uridine transmembrane transporter activity / Azathioprine ADME / brush border membrane / plasma membrane
Similarity search - Function
Concentrative nucleoside transporter N-terminal domain / Concentrative nucleoside transporter / Concentrative nucleoside transporter C-terminal domain / Concentrative nucleoside transporter, metazoan/bacterial / Na+ dependent nucleoside transporter N-terminus / Na+ dependent nucleoside transporter C-terminus / Nucleoside transporter/FeoB GTPase, Gate domain / Nucleoside recognition
Similarity search - Domain/homology
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / : / Sodium/nucleoside cotransporter
Similarity search - Component
Biological speciesBos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsWright, N.J. / Lee, S.-Y.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R21AI166134 United States
CitationJournal: Nat.Chem.Biol. / Year: 2024
Title: Antiviral drug recognition and elevator-type transport motions of CNT3.
Authors: Wright, N.J. / Zhang, F. / Suo, Y. / Kong, L. / Yin, Y. / Fedor, J.G. / Sharma, K. / Borgnia, M.J. / Im, W. / Lee, S.Y.
History
DepositionAug 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.2Sep 18, 2024Group: Data collection / Database references / Category: citation_author / em_admin
Item: _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium/nucleoside cotransporter
B: Sodium/nucleoside cotransporter
C: Sodium/nucleoside cotransporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)248,10121
Polymers238,1303
Non-polymers9,97118
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sodium/nucleoside cotransporter


Mass: 79376.773 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: SLC28A3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F1MGR1
#2: Chemical ChemComp-U7I / 2'-deoxy-2'-fluoro-2'-methyluridine / PSI-6206


Mass: 260.219 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H13FN2O5 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na
#4: Chemical
ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: bCNT3 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.232 MDa / Experimental value: YES
Source (natural)Organism: Bos taurus (domestic cattle)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: sf9
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
1150 mMNaCl1
220 mMTris1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3820
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3particle selection
2Latitude3.5image acquisition
4CTFFIND4CTF correction
7Coot0.96model fitting
9PHENIX1.18model refinement
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
12cryoSPARC3.3classification
13cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2827294
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199596 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3TIJ

3tij


Accession code: 3TIJ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413005
ELECTRON MICROSCOPYf_angle_d0.49917634
ELECTRON MICROSCOPYf_dihedral_angle_d16.2754464
ELECTRON MICROSCOPYf_chiral_restr0.0382007
ELECTRON MICROSCOPYf_plane_restr0.0032118

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