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- EMDB-41745: Cryo-EM reconstruction of bovine concentrative nucleoside transpo... -

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Basic information

Entry
Database: EMDB / ID: EMD-41745
TitleCryo-EM reconstruction of bovine concentrative nucleoside transporter 3 in complex with Molnupiravir, condition 1, INT3-INT3-OFS conformation
Map datafull map
Sample
  • Complex: bCNT3 trimer
    • Protein or peptide: bCNT3
Keywordsmembrane protein / transporter / nucleoside / TRANSPORT PROTEIN
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 / uridine transmembrane transporter activity / purine nucleoside transmembrane transport / 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
Sodium/nucleoside cotransporter
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.28 Å
AuthorsWright NJ / Lee S-Y
Funding support United States, 1 items
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: Nicholas J Wright / Feng Zhang / Yang Suo / Lingyang Kong / Ying Yin / Justin G Fedor / Kedar Sharma / Mario J Borgnia / Wonpil Im / Seok-Yong Lee /
Abstract: Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human ...Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.
History
DepositionAug 26, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41745.png

Downloads & links

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Map

FileDownload / File: emd_41745.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.081.081.08
CCP4 map header1.881.881.88
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.56182295 - 1.0826643
Average (Standard dev.)0.0030957768 (±0.048389085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map 2

Fileemd_41745_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : bCNT3 trimer

EntireName: bCNT3 trimer
Components
  • Complex: bCNT3 trimer
    • Protein or peptide: bCNT3

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Supramolecule #1: bCNT3 trimer

SupramoleculeName: bCNT3 trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 232 KDa

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Macromolecule #1: bCNT3

MacromoleculeName: bCNT3 / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDYKDDDDKL EATMAMSSKI SVELQRVAAL PAQGCSNTGF QNDEDGFENQ NPSGNDHSLR NRVVQNREHE NGKQVEEHIT IGQDSLRKDE EEEDDQETHR KGCLERMCGR MSDFCREHKT TLRYIIWGIL IAGYLALVIA ACVMNFHRAL PLFVITVVAI FFVVWDHLMA ...String:
MDYKDDDDKL EATMAMSSKI SVELQRVAAL PAQGCSNTGF QNDEDGFENQ NPSGNDHSLR NRVVQNREHE NGKQVEEHIT IGQDSLRKDE EEEDDQETHR KGCLERMCGR MSDFCREHKT TLRYIIWGIL IAGYLALVIA ACVMNFHRAL PLFVITVVAI FFVVWDHLMA KYESQIARFL SPGQRLLDSH WFWLKWVIWG CLILGVILWL VFDTAKLGQQ QLVSFGGLII YTSLTFLFSK HPTKVYWRPV FWGIGLQFLL GLLILRTEPG FMAFDWLGKQ VQTFLGYSDA GASFVFGEKY TDHFFAFKVL PIVIFFSTVM SMLYYLGLMQ WIIRKVGWVM LVTMGTSPVE SVVASGNIFI GQTESPLLVR PYLPYVTKSE LHAIMTAGFS TIAGSVLGAY ISFGVSSSHL LTASVMSAPA ALAISKLFWP ETETPKINLK NAMKMESGDS RNLLEAATQG ASSSISLVAN IAVNLIAFLA LLSFMNSALS WLGNMFDYPQ LSFEVICSYV FMPFAFMMGV DWQDSFMVAK LIGYKTFFNE FVAYQQLSKL ISLRQVGGPK FVDGVQQYMS MRSEAISTYA LCGFANFGSL GIVIGGLTSM APSRKRDITA GAMRALIAGT IACFLTACIA GMLTNTPVDI NCHHILENAF NSGLVRNTTN VVSCCQGLLS SAVVKGPGEV IPTGNHSLYS LKNCCNLLNT PTLNCSWIPN VLSNS

UniProtKB: Sodium/nucleoside cotransporter

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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

Concentration8 mg/mL
BufferpH: 8 / Component:
ConcentrationFormula
150.0 mMNaCl
20.0 mMTris
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 4715 / Average exposure time: 2.4 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2592142
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 8163
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3)

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Atomic model buiding 1

Initial modelPDB ID:

3tij


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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