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- PDB-8dep: Cryo-EM structure of the human reduced folate carrier, apo condition -

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Basic information

Entry
Database: PDB / ID: 8dep
TitleCryo-EM structure of the human reduced folate carrier, apo condition
ComponentsReduced folate transporter,Soluble cytochrome b562
KeywordsTRANSPORT PROTEIN / reduced folate carrier / membrane protein / membrane transporter / methotrexate / SLC19A1 / solute carrier family 19
Function / homology
Function and homology information


folate:monoatomic anion antiporter activity / methotrexate transport / folic acid transmembrane transporter activity / methotrexate transmembrane transporter activity / folic acid transport / folate transmembrane transport / folate import across plasma membrane / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / 2',3'-cyclic GMP-AMP binding ...folate:monoatomic anion antiporter activity / methotrexate transport / folic acid transmembrane transporter activity / methotrexate transmembrane transporter activity / folic acid transport / folate transmembrane transport / folate import across plasma membrane / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / 2',3'-cyclic GMP-AMP binding / Metabolism of folate and pterines / xenobiotic transmembrane transport / organic anion transport / positive regulation of cGAS/STING signaling pathway / : / folic acid binding / antiporter activity / folic acid metabolic process / xenobiotic transmembrane transporter activity / transport across blood-brain barrier / brush border membrane / female pregnancy / electron transport chain / response to toxic substance / basolateral plasma membrane / periplasmic space / electron transfer activity / apical plasma membrane / iron ion binding / response to xenobiotic stimulus / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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)GM137421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129547 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
DepositionJun 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 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 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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: electron microscopy
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
Source method: isolated from a genetically manipulated source
Details: UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome ...Details: UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562,UNPID P41440 residues 215-241 removed and replaced with a fragment of soluble cytochrome b562
Source: (gene. exp.) Homo sapiens (human) / 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 / Type: COMPLEX / Details: apo state / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
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: 2.3 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12201
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
4cryoSPARC3CTF correction
5CTFFIND4CTF correction
8Coot0.88model fitting
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
14PHENIX1.13-2998model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2536392
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138522 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 75 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7XT7

7xt7


Pdb chain-ID: A / Accession code: 7XT7 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033333
ELECTRON MICROSCOPYf_angle_d0.9664615
ELECTRON MICROSCOPYf_dihedral_angle_d12.2041046
ELECTRON MICROSCOPYf_chiral_restr0.048582
ELECTRON MICROSCOPYf_plane_restr0.005521

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