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- EMDB-28587: Cryo-EM structure of the organic cation transporter 1 in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-28587
TitleCryo-EM structure of the organic cation transporter 1 in complex with diphenhydramine
Map datafull map
Sample
  • Complex: OCT1
    • Protein or peptide: OCT1
  • Ligand: N-[2-(BENZHYDRYLOXY)ETHYL]-N,N-DIMETHYLAMINE
KeywordsOrganic cation / transport / membrane protein / TRANSPORT PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsSuo Y / Wright NJ / Lee S-Y
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM137421 United States
CitationJournal: bioRxiv / Year: 2023
Title: Molecular basis of polyspecific drug binding and transport by OCT1 and OCT2.
Authors: Yang Suo / Nicholas J Wright / Hugo Guterres / Justin G Fedor / Kevin John Butay / Mario J Borgnia / Wonpil Im / Seok-Yong Lee /
Abstract: A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition . In mammals, organic cation transporter subtypes 1 ...A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition . In mammals, organic cation transporter subtypes 1 and 2 (OCT1 and OCT2, also known as SLC22A1 and SLC22A2, respectively) are polyspecific transporters responsible for the uptake and clearance of structurally diverse cationic compounds in the liver and kidneys, respectively . Notably, it is well established that human OCT1 and OCT2 play central roles in the pharmacokinetics, pharmacodynamics, and drug-drug interactions (DDI) of many prescription medications, including metformin . Despite their importance, the basis of polyspecific cationic drug recognition and the alternating access mechanism for OCTs have remained a mystery. Here, we present four cryo-EM structures of apo, substrate-bound, and drug-bound OCT1 and OCT2 in outward-facing and outward-occluded states. Together with functional experiments, docking, and molecular dynamics simulations, these structures uncover general principles of organic cation recognition by OCTs and illuminate unexpected features of the OCT alternating access mechanism. Our findings set the stage for a comprehensive structure-based understanding of OCT-mediated DDI, which will prove critical in the preclinical evaluation of emerging therapeutics.
History
DepositionOct 16, 2022-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28587.png

Downloads & links

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Map

FileDownload / File: emd_28587.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy EMDB: 0.3
Minimum - Maximum-1.0133756 - 1.8908701
Average (Standard dev.)0.0032157507 (±0.047972564)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 216.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map 1

Fileemd_28587_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_28587_half_map_2.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 : OCT1

EntireName: OCT1
Components
  • Complex: OCT1
    • Protein or peptide: OCT1
  • Ligand: N-[2-(BENZHYDRYLOXY)ETHYL]-N,N-DIMETHYLAMINE

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Supramolecule #1: OCT1

SupramoleculeName: OCT1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60 kDa/nm

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Macromolecule #1: OCT1

MacromoleculeName: OCT1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.299562 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPTVDDVLEQ VGEFGWFQKQ AFLTLCLLSA AFAPIYVGIV FLGFTPDHRC RSPGVAELSQ RCGWSLAEEL NYTVPGLGAA GEAFPRQCR RYEVDWNQSA LSCVDPLASL AANRSHLPLG PCQHGWVYDT PGSSIVTEFN LVCADSWKVD LFQSCVNVGF F LGSLGVGY ...String:
MPTVDDVLEQ VGEFGWFQKQ AFLTLCLLSA AFAPIYVGIV FLGFTPDHRC RSPGVAELSQ RCGWSLAEEL NYTVPGLGAA GEAFPRQCR RYEVDWNQSA LSCVDPLASL AANRSHLPLG PCQHGWVYDT PGSSIVTEFN LVCADSWKVD LFQSCVNVGF F LGSLGVGY IADRFGRKLC LLATTLISAV SGVLMAVAPD YTSMLLFRLL QGLVSKGSWM SGYTLITEFV GSGYRRTVAI LY QMAFTVG LVLLSGVAYA IPHWRWLQLA VSLPTFLFLL YYWCVPESPR WLLSQKRNTQ AIKIMDHIAQ KNGKLPPADL KML SLKEDS TEKLSPSFAD LFRTPQLRKH TFILMYLWFT SSVLYQGLIM HMGATGGNLY LDFFYSALVE FPAAFIILVT IDRV GRIYP LAVSNLVAGA ACLIMIFISQ DLHWLNITVA CVGRMGITIV FQMVCLVNAE LYPTFIRNLG VMVCSSLCDL GGIIT PFLV FRLMEVWQGL PLILFTVVGL VAGGMTLLLP ETKGVALPET IEDAENLGRK AKPKENTIYL QVQTSELPGT

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Macromolecule #3: N-[2-(BENZHYDRYLOXY)ETHYL]-N,N-DIMETHYLAMINE

MacromoleculeName: N-[2-(BENZHYDRYLOXY)ETHYL]-N,N-DIMETHYLAMINE / type: ligand / ID: 3 / Number of copies: 1 / Formula: 2PM
Molecular weightTheoretical: 255.355 Da
Chemical component information

ChemComp-2PM:
N-[2-(BENZHYDRYLOXY)ETHYL]-N,N-DIMETHYLAMINE / Diphenhydramine

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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

Concentration4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
150.0 mMNaClSodium chlorideSodium Chloride
0.06 %DigitoninDigitonin
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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: 7145 / Average exposure time: 3.7 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 693720
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 189193

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 164.4
Output model

PDB-8et7:
Cryo-EM structure of the organic cation transporter 1 in complex with diphenhydramine

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