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- EMDB-26155: Cryo-EM structure of the human reduced folate carrier in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26155
TitleCryo-EM structure of the human reduced folate carrier in complex with methotrexate
Map datasharpened full map
Sample
  • Complex: Human reduced folate carrier SLC19A1
    • Protein or peptide: Reduced folate transporter,Soluble cytochrome b562
  • Ligand: METHOTREXATE
  • Ligand: Digitonin
Keywordstransporter / reduced folate carrier / SLC19A1 / TRANSPORT PROTEIN / anion exchanger
Function / homology
Function and homology information


folic acid transmembrane transporter activity / folate:monoatomic anion antiporter activity / methotrexate transport / folate transmembrane transport / methotrexate transmembrane transporter activity / folic acid transport / folate import across plasma membrane / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / organic anion transport ...folic acid transmembrane transporter activity / folate:monoatomic anion antiporter activity / methotrexate transport / folate transmembrane transport / methotrexate transmembrane transporter activity / folic acid transport / folate import across plasma membrane / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / organic anion transport / 2',3'-cyclic GMP-AMP binding / xenobiotic transmembrane transport / organic anion transmembrane transporter activity / Metabolism of folate and pterines / antiporter activity / folic acid binding / folic acid metabolic process / xenobiotic transmembrane transporter activity / transport across blood-brain barrier / female pregnancy / brush border membrane / basolateral plasma membrane / electron transfer activity / periplasmic space / iron ion binding / apical plasma membrane / heme binding / plasma membrane
Similarity search - Function
Reduced folate transporter SLC19A1 / Reduced folate carrier / Reduced folate carrier / MFS transporter superfamily / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562
Similarity search - Domain/homology
Soluble cytochrome b562 / Reduced folate transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWright NJ / Fedor JG / Lee S-Y
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM137421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
American Heart Association20PRE35210058 United States
CitationJournal: Nature / Year: 2022
Title: Methotrexate recognition by the human reduced folate carrier SLC19A1.
Authors: Nicholas J Wright / Justin G Fedor / Han Zhang / Pyeonghwa Jeong / Yang Suo / Jiho Yoo / Jiyong Hong / Wonpil Im / Seok-Yong Lee /
Abstract: Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during ...Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell, as well as chemotherapeutic agents such as methotrexate. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.
History
DepositionFeb 7, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26155.png

Downloads & links

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Map

FileDownload / File: emd_26155.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened full map
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.71251464 - 1.6208541
Average (Standard dev.)0.002196488 (±0.033134315)
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 B

Fileemd_26155_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_26155_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human reduced folate carrier SLC19A1

EntireName: Human reduced folate carrier SLC19A1
Components
  • Complex: Human reduced folate carrier SLC19A1
    • Protein or peptide: Reduced folate transporter,Soluble cytochrome b562
  • Ligand: METHOTREXATE
  • Ligand: Digitonin

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Supramolecule #1: Human reduced folate carrier SLC19A1

SupramoleculeName: Human reduced folate carrier SLC19A1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Covalently modified Lys411 with methotrexate
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Reduced folate transporter,Soluble cytochrome b562

MacromoleculeName: Reduced folate transporter,Soluble cytochrome b562 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 74.797195 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVPSSPAVEK QVPVEPGPDP ELRSWRHLVC YLCFYGFMAQ IRPGESFITP YLLGPDKNFT REQVTNEITP VLSYSYLAVL VPVFLLTDY LRYTPVLLLQ GLSFVSVWLL LLLGHSVAHM QLMELFYSVT MAARIAYSSY IFSLVRPARY QRVAGYSRAA V LLGVFTSS ...String:
MVPSSPAVEK QVPVEPGPDP ELRSWRHLVC YLCFYGFMAQ IRPGESFITP YLLGPDKNFT REQVTNEITP VLSYSYLAVL VPVFLLTDY LRYTPVLLLQ GLSFVSVWLL LLLGHSVAHM QLMELFYSVT MAARIAYSSY IFSLVRPARY QRVAGYSRAA V LLGVFTSS VLGQLLVTVG RVSFSTLNYI SLAFLTFSVV LALFLKRPKR SLFFNRDLED NWETLNDNLK VIEKADNAAQ VK DALTKMR AAALDAQKAT PPKLEDKSPD SPEMKDFRHG FDILVGQIDD ALKLANEGKV KEAQAAAEQL KTTRNAYIQK YLL RVACGD SVLARMLREL GDSLRRPQLR LWSLWWVFNS AGYYLVVYYV HILWNEVDPT TNSARVYNGA ADAASTLLGA ITSF AAGFV KIRWARWSKL LIAGVTATQA GLVFLLAHTR HPSSIWLCYA AFVLFRGSYQ FLVPIATFQI ASSLSKELCA LVFGV NTFF ATIVKTIITF IVSDVRGLGL PVRKQFQLYS VYFLILSIIY FLGAMLDGLR HCQRGHHPRQ PPAQGLRSAA EEKAAQ ALS VQDKGLGGLQ PAQSPPLSPE DSLGAVGPAS LEQRQSDPYL AQAPAPQAAE FLSPVTTPSP CTLCSAQASG PEAADET CP QLAVHPPGVS KLGLQCLPSD GVQNVNQANS LEVLFQ

UniProtKB: Reduced folate transporter, Soluble cytochrome b562, Reduced folate transporter

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Macromolecule #2: METHOTREXATE

MacromoleculeName: METHOTREXATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: MTX
Molecular weightTheoretical: 454.439 Da
Chemical component information

ChemComp-MTX:
METHOTREXATE / chemotherapy*YM / Methotrexate

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Macromolecule #3: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 3 / Number of copies: 1 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM / Digitonin

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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

Concentration5 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 200 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.00039000000000000005 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 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) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 5 / Number real images: 25507 / Average exposure time: 4.6 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: 9098387
Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3)
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 492117

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 53.6
Output model

PDB-7tx6:
Cryo-EM structure of the human reduced folate carrier in complex with methotrexate

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