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- PDB-7bc7: Cryo-EM structure of the proton coupled folate transporter at pH ... -

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Basic information

Entry
Database: PDB / ID: 7bc7
TitleCryo-EM structure of the proton coupled folate transporter at pH 6.0 bound to pemetrexed
Components
  • Proton-coupled folate transporter
  • nanobody
KeywordsMEMBRANE PROTEIN / transporter folate proton symporter
Function / homology
Function and homology information


folic acid:proton symporter activity / Metabolism of folate and pterines / Heme signaling / Iron uptake and transport / methotrexate transmembrane transporter activity / folate import across plasma membrane / folic acid binding / transmembrane transporter activity / transmembrane transport / basolateral plasma membrane ...folic acid:proton symporter activity / Metabolism of folate and pterines / Heme signaling / Iron uptake and transport / methotrexate transmembrane transporter activity / folate import across plasma membrane / folic acid binding / transmembrane transporter activity / transmembrane transport / basolateral plasma membrane / endosome membrane / endosome / apical plasma membrane / membrane / plasma membrane
Similarity search - Function
Major facilitator superfamily / Major Facilitator Superfamily / MFS transporter superfamily
Similarity search - Domain/homology
Chem-LYA / Proton-coupled folate transporter
Similarity search - Component
Biological speciesGallus gallus (chicken)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsParker, J.L. / Deme, J.C. / Lea, S.M. / Newstead, S.
Funding support United Kingdom, 10items
OrganizationGrant numberCountry
Wellcome Trust210536 United Kingdom
Wolfson FoundationWL160052 United Kingdom
Wellcome Trust209194 United Kingdom
Wellcome Trust100298 United Kingdom
Engineering and Physical Sciences Research CouncilEP/L000253/1 United Kingdom
Wellcome Trust219531 United Kingdom
Wellcome Trust215519 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/M011984/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/S021043/1 United Kingdom
Wellcome Trust203741 United Kingdom
CitationJournal: Nature / Year: 2021
Title: Structural basis of antifolate recognition and transport by PCFT.
Authors: Joanne L Parker / Justin C Deme / Gabriel Kuteyi / Zhiyi Wu / Jiandong Huo / I David Goldman / Raymond J Owens / Philip C Biggin / Susan M Lea / Simon Newstead /
Abstract: Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S- ...Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine. Folate deficiency is associated with a number of developmental, immune and neurological disorders. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus, and is an important route for the delivery of antifolate drugs in cancer chemotherapy. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.
History
DepositionDec 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 12, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 9, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 7, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jul 14, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.4Aug 11, 2021Group: Database references / Category: citation_author / database_2
Item: _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Proton-coupled folate transporter
B: nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,2543
Polymers64,8272
Non-polymers4271
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3090 Å2
ΔGint-11 kcal/mol
Surface area22480 Å2
MethodPISA

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Components

#1: Protein Proton-coupled folate transporter


Mass: 51366.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: PCFT / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: E6Y8U5
#2: Antibody nanobody


Mass: 13460.006 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Lama glama (llama)
#3: Chemical ChemComp-LYA / 2-{4-[2-(2-AMINO-4-OXO-4,7-DIHYDRO-3H-PYRROLO[2,3-D]PYRIMIDIN-5-YL)-ETHYL]-BENZOYLAMINO}-PENTANEDIOIC ACID / LY231514


Mass: 427.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H21N5O6 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, chemotherapy*YM
Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of the proton coupled folate transporter at pH 6.0 bound to pemetrexedCOMPLEX#1-#20MULTIPLE SOURCES
2proton-coupled folate transporterCOMPLEX#11RECOMBINANT
3nanobodyCOMPLEX#21NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.059331 MDaNO
220.046399 MDaNO
330.012932 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Gallus gallus (chicken)9031
23Lama glama (llama)9844
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 58.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 341318 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0084311
ELECTRON MICROSCOPYf_angle_d0.8375873
ELECTRON MICROSCOPYf_dihedral_angle_d20.005610
ELECTRON MICROSCOPYf_chiral_restr0.051672
ELECTRON MICROSCOPYf_plane_restr0.005733

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