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- PDB-9sqh: Cryo-EM structure of the Arabidopsis thaliana CAT4 transporter in... -

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Basic information

Entry
Database: PDB / ID: 9sqh
TitleCryo-EM structure of the Arabidopsis thaliana CAT4 transporter in the outward-open apo state (without synthetic nanobody)
ComponentsCationic amino acid transporter 4, vacuolar
KeywordsTRANSPORT PROTEIN / cationic amino acid transporter / APC fold / LeuT fold / cholesterol / outward-open state / cationic amino acids / SLC7 / MEMBRANE PROTEIN
Function / homologyCationic amino acid transporter, C-terminal / C-terminus of AA_permease / plant-type vacuole membrane / Amino acid/polyamine transporter I / Amino acid permease / plant-type vacuole / amino acid transmembrane transporter activity / amino acid transport / Cationic amino acid transporter 4, vacuolar
Function and homology information
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.23 Å
AuthorsKolokouris, D. / Zeng, Y.C. / Newstead, S.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011224/1 United Kingdom
CitationJournal: Nat Commun / Year: 2026
Title: Structural basis for pH-responsive amino acid transport via SLC7A4.
Authors: Dimitrios Kolokouris / Anuja Bothra / Takafumi Kato / Yi C Zeng / Simon Lichtinger / Joanne L Parker / Philip C Biggin / Simon Newstead /
Abstract: The transport of amino acids across cell membranes is essential for metabolism, neuronal signalling, and immune system function. The amino acid polyamine organocation (APC) superfamily controls amino ...The transport of amino acids across cell membranes is essential for metabolism, neuronal signalling, and immune system function. The amino acid polyamine organocation (APC) superfamily controls amino acid transport via mechanisms including amino acid exchange, facilitative diffusion, and sodium- or proton-coupled transport. Although many mammalian APC members functioning as exchangers and sodium-coupled systems have been identified, the mechanisms underlying pH-regulated amino acid transport in mammalian cells remain unclear. Here, we show that the plasma membrane amino acid transporter SLC7A4 is regulated by low extracellular pH and functions as a leucine transporter in human cells. Using Cryo-EM structures of the plant homologue, CAT4, from Arabidopsis thaliana in outward-open apo and L-ornithine-bound states, as well as transport assays and molecular dynamics simulations based on homology models of the human transporter, we identify residues responsible for amino acid selectivity that supports an allosteric mechanism linking ligand recognition to pH regulation. This mechanism is consistent with an evolutionary link to proton-coupled prokaryotic homologues. Overall, our findings provide a structural and functional basis for pH-gated leucine transport by the human SLC7A4 transporter and provides a framework for understanding amino acid selectivity within the wider SLC7 family.
History
DepositionSep 22, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 8, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cationic amino acid transporter 4, vacuolar


Theoretical massNumber of molelcules
Total (without water)63,6801
Polymers63,6801
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cationic amino acid transporter 4, vacuolar


Mass: 63680.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: CAT4, At3g03720, F20H23.25 / Plasmid: pDDGFP2-LEU2d / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): BJ5460 / References: UniProt: Q8W4K3
Has protein modificationN

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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: Arabidopsis thaliana cationic amino acid transporter 4 solubilised in LMNG
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pDDGFP-Leu2d
Buffer solutionpH: 7.5 / Details: 20 mM Tris-HCl, 150 mM NaCl, 0.003% w/v LMNG
SpecimenConc.: 4.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 39.9 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 30514

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIX1.21.1_5286model refinement
14ISOLDE1.8model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8226459
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120868 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 9HJK

9hjk


Accession code: 9HJK / Details: Chain A of 9HJK used for initial fitting / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 77.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023255
ELECTRON MICROSCOPYf_angle_d0.51634442
ELECTRON MICROSCOPYf_chiral_restr0.0365567
ELECTRON MICROSCOPYf_plane_restr0.0033529
ELECTRON MICROSCOPYf_dihedral_angle_d4.1191454

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