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- EMDB-12140: Cryo-EM structure of the outward open proton coupled folate trans... -

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Basic information

Entry
Database: EMDB / ID: EMD-12140
TitleCryo-EM structure of the outward open proton coupled folate transporter at pH 7.5
Map dataglobally sharpened map from cryosparc non uniform refinement
Sample
  • Complex: Complex of the outward open proton coupled folate transporter with nanobody at pH 7.5
    • Complex: proton-coupled folate transporter
      • Protein or peptide: Proton-coupled folate transporter
    • Complex: nanobodySingle-domain antibody
      • Protein or peptide: nanobodySingle-domain antibody
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
Proton-coupled folate transporter
Similarity search - Component
Biological speciesGallus gallus (chicken) / Lama glama (llama) / Llama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsParker JL / Deme JC / Lea SM / Newstead S
Funding support United Kingdom, 10 items
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, 2020-
Header (metadata) releaseMay 12, 2021-
Map releaseMay 12, 2021-
UpdateAug 11, 2021-
Current statusAug 11, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bc6
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12140.png

Downloads & links

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Map

FileDownload / File: emd_12140.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationglobally sharpened map from cryosparc non uniform refinement
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.7
Minimum - Maximum-4.4268737 - 5.9175243
Average (Standard dev.)0.0005494213 (±0.09975655)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-4.4275.9180.001

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Supplemental data

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Mask #1

Fileemd_12140_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map from cryosparc non uniform refinement

Fileemd_12140_additional_1.map
Annotationunsharpened map from cryosparc non uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B from cryosparc non uniform refinement

Fileemd_12140_half_map_1.map
Annotationhalf map B from cryosparc non uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A from cryosparc non uniform refinement

Fileemd_12140_half_map_2.map
Annotationhalf map A from cryosparc non uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the outward open proton coupled folate transporter wit...

EntireName: Complex of the outward open proton coupled folate transporter with nanobody at pH 7.5
Components
  • Complex: Complex of the outward open proton coupled folate transporter with nanobody at pH 7.5
    • Complex: proton-coupled folate transporter
      • Protein or peptide: Proton-coupled folate transporter
    • Complex: nanobodySingle-domain antibody
      • Protein or peptide: nanobodySingle-domain antibody

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Supramolecule #1: Complex of the outward open proton coupled folate transporter wit...

SupramoleculeName: Complex of the outward open proton coupled folate transporter with nanobody at pH 7.5
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: proton-coupled folate transporter

SupramoleculeName: proton-coupled folate transporter / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: nanobody

SupramoleculeName: nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Proton-coupled folate transporter

MacromoleculeName: Proton-coupled folate transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 51.36657 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAAPSDPPTA ATPPAPPPPA RRCLPAPSVE PLLFLATLAL GLQVPLATQY LWDRLGAERG YVGPNASSPH GCGNGSGAVD PLREEVEAL VAHWNLCINL GGFFVGLFSV TLFGPWSDSV GRRPVLVLPA VGMAVQAAVY LLVMYLRLHV AYLLLGRIIS G LLGDYNLI ...String:
MAAPSDPPTA ATPPAPPPPA RRCLPAPSVE PLLFLATLAL GLQVPLATQY LWDRLGAERG YVGPNASSPH GCGNGSGAVD PLREEVEAL VAHWNLCINL GGFFVGLFSV TLFGPWSDSV GRRPVLVLPA VGMAVQAAVY LLVMYLRLHV AYLLLGRIIS G LLGDYNLI LAGCFASVAD SSNQRTRTFR VAILEACLGV AGMVASVGGG QWRKAEGYIN PFWLVLAASL AAALYAALCL QE TVKQRRA AKLLTLQHYK AVYKLYTAPE DLSSRRKLAL YSLAFFLLVT VHFGTKDLYV LYELGSPLCW ASDLIGYGSA ASY LAYLSS LGGLRLLQLC LEDTWVAEIG LISNIAGLVV ISLATTTPLM FTGYGIMFLS MAATPVIRAK LSKLVSETEQ GALF ASVAC VEGLCSLVAT GVFNSLYPST LHFMRGFPFL FGAILLLIPA AIMGWIEIQD SNLQYSHFSD ASSSPADGGE NLYFQ

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Macromolecule #2: nanobody

MacromoleculeName: nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Llama (llama)
Molecular weightTheoretical: 13.460006 KDa
SequenceString:
GPSQVQLVES GGGLVQPGGS LRLSCAASGF TFSRYWMYWV RQAPGKGPEW LSHMNPSGSD IKYTDSVKGR FTISRDNAKN TLYLQMNSL KPDDTAVYYC VADRRALGSP EYWGQGTQVT VSSA

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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: 7.5
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 59.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 174399
FSC plot (resolution estimation)

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