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- PDB-7tx7: Cryo-EM structure of the human reduced folate carrier -

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Basic information

Entry
Database: PDB / ID: 7tx7
TitleCryo-EM structure of the human reduced folate carrier
ComponentsReduced folate transporter,Soluble cytochrome b562
KeywordsTRANSPORT PROTEIN / Membrane transporter / reduced folate carrier / SLC19 / SLC19A1 / anion exchanger
Function / homology
Function and homology information


folic acid transmembrane transporter activity / folate:monoatomic anion antiporter activity / methotrexate transport / methotrexate transmembrane transporter activity / folic acid transport / folate transmembrane transport / folate import across plasma membrane / positive regulation of cGAS/STING signaling pathway / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane ...folic acid transmembrane transporter activity / folate:monoatomic anion antiporter activity / methotrexate transport / methotrexate transmembrane transporter activity / folic acid transport / folate transmembrane transport / folate import across plasma membrane / positive regulation of cGAS/STING signaling pathway / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / 2',3'-cyclic GMP-AMP binding / organic anion transport / 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 / electron transport chain / basolateral plasma membrane / periplasmic space / electron transfer activity / iron ion binding / apical plasma membrane / heme binding / plasma membrane
Similarity search - Function
Reduced folate transporter SLC19A1 / Reduced folate carrier / Reduced folate carrier / Cytochrome b562 / Cytochrome b562 / MFS transporter superfamily / Cytochrome c/b562
Similarity search - Domain/homology
Soluble cytochrome b562 / Reduced folate transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWright, N.J. / Fedor, J.G. / Lee, S.-Y.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 2.0Oct 5, 2022Group: Advisory / Database references ...Advisory / Database references / Polymer sequence / Source and taxonomy / Structure summary
Category: entity / entity_poly ...entity / entity_poly / entity_poly_seq / entity_src_gen / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_residues / struct_ref_seq_dif
Item: _entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code ..._entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_end_seq_num
Revision 2.1Oct 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 2.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Reduced folate transporter,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,7145
Polymers74,7971
Non-polymers4,9174
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area20380 Å2
MethodPISA

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Components

#1: Protein Reduced folate transporter,Soluble cytochrome b562 / FOLT / Intestinal folate carrier 1 / IFC-1 / Placental folate transporter / Reduced folate carrier ...FOLT / Intestinal folate carrier 1 / IFC-1 / Placental folate transporter / Reduced folate carrier protein / RFC / hRFC / Reduced folate transporter 1 / RFT-1 / Solute carrier family 19 member 1 / hSLC19A1 / Cytochrome b-562


Mass: 74797.195 Da / Num. of mol.: 1
Mutation: UNP residues 215-241 replaced with engineered epitope
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: SLC19A1, FLOT1, RFC1, cybC / Cell line (production host): HEK293S GnTi- / Production host: Homo sapiens (human) / References: UniProt: P41440, UniProt: P0ABE7
#2: Chemical
ChemComp-AJP / Digitonin


Mass: 1229.312 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM
Has ligand of interestN

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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: human reduced folate carrier SLC19A1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Escherichia coli (E. coli)562
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTi-
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 85 % / 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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.6 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 21002
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2Latitudeimage acquisition
4cryoSPARC3CTF correction
5CTFFIND4CTF correction
8Coot0.88model fitting
10RELION3.1initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
20PHENIX1.13-2998model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4538955
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 298876 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 120 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033310
ELECTRON MICROSCOPYf_angle_d0.9484583
ELECTRON MICROSCOPYf_dihedral_angle_d11.1311041
ELECTRON MICROSCOPYf_chiral_restr0.047581
ELECTRON MICROSCOPYf_plane_restr0.005518

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