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- EMDB-28589: Cryo-EM structure of the organic cation transporter 2 in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-28589
TitleCryo-EM structure of the organic cation transporter 2 in complex with 1-methyl-4-phenylpyridinium
Map datamain map
Sample
  • Complex: OCT2
    • Protein or peptide: OCT2
  • Ligand: 1-methyl-4-phenylpyridin-1-ium
KeywordsCation / transport / membrane protein / TRANSPORT PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.61 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-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28589.png

Downloads & links

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Map

FileDownload / File: emd_28589.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 200 pix.
= 216. Å		1.08 Å/pix.
x 200 pix.
= 216. Å		1.08 Å/pix.
x 200 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.15
Minimum - Maximum-1.8743418 - 2.434535
Average (Standard dev.)0.002525262 (±0.04747627)
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 1

Fileemd_28589_half_map_1.map
Annotationhalf 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half 2

Fileemd_28589_half_map_2.map
Annotationhalf 2
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : OCT2

EntireName: OCT2
Components
  • Complex: OCT2
    • Protein or peptide: OCT2
  • Ligand: 1-methyl-4-phenylpyridin-1-ium

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Supramolecule #1: OCT2

SupramoleculeName: OCT2 / 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: OCT2

MacromoleculeName: OCT2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 62.030547 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPTTFDDILE HIGEFGRFQK QTFFLLCLLS AAFAPIYVGI VFLGFTPDHR CRSPGVAELS QRCGWSLEEE LNYTVPGVGS SGEASPSQC RRYEVDWNQT GLSCTDPLAS LAANRSHLPL GPCQDGWVYD TPGSSIVTEF NLVCADSWML DLVQAAVNVG F FVGSMSIG ...String:
MPTTFDDILE HIGEFGRFQK QTFFLLCLLS AAFAPIYVGI VFLGFTPDHR CRSPGVAELS QRCGWSLEEE LNYTVPGVGS SGEASPSQC RRYEVDWNQT GLSCTDPLAS LAANRSHLPL GPCQDGWVYD TPGSSIVTEF NLVCADSWML DLVQAAVNVG F FVGSMSIG YIADRFGRKL CLLVTILINA ISGVLMAVAP NYTWMLIFRL IQGLVSKGGW LIGYTLPTEF VGLEYRRTVG IL YQMAFSV GLLVLAGVAY AIPHWRWLQL AVTLPNFFFL LYYWCIPESP RWLISQNKNA KAMKIIKHIA KKNGKKLPKS LQE ERKETE VGEKLNPSFL DLVRTPQIRK HTLILMYNWF TSSVLYQGLI MHMGIAGGNI YLDFFYSALV EFPAAFIIIL TIDR IGRRY PWAAANMVAG AACLITAFIP DGLFWLKTTV ACLGRMGITM AFEMVCFVNT ELYPTFIRNL GVLVCSSLCD IGGII TPFL VYRLASIWLE LPLVVFAVLG LIAGGLVLLL PETKGKPLPE TIEDAENPHR PRKNKEKMIY LQVLLSDIPD N

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Macromolecule #2: 1-methyl-4-phenylpyridin-1-ium

MacromoleculeName: 1-methyl-4-phenylpyridin-1-ium / type: ligand / ID: 2 / Number of copies: 1 / Formula: WRF
Molecular weightTheoretical: 170.23 Da
Chemical component information

ChemComp-WRF:
1-methyl-4-phenylpyridin-1-ium

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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: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
150.0 mMNaClSodium Chloride
0.06 %DigitoninDigitonin
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 240 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / 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: 8029 / Average exposure time: 3.7 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 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: 1141906
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 73474
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.2)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 141.9
Output model

PDB-8et9:
Cryo-EM structure of the organic cation transporter 2 in complex with 1-methyl-4-phenylpyridinium

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