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- PDB-9sp8: Cryo-EM structure of the Arabidopsis thaliana CAT4 transporter in... -

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Basic information

Entry
Database: PDB / ID: 9sp8
TitleCryo-EM structure of the Arabidopsis thaliana CAT4 transporter in the outward-open L-ornithine bound state
Components
  • Cationic amino acid transporter 4, vacuolar
  • SybB5
KeywordsTRANSPORT PROTEIN / membrane transporter / cationic amino acid
Function / homology
Function and homology information


plant-type vacuole membrane / plant-type vacuole / amino acid transmembrane transporter activity / amino acid transport
Similarity search - Function
Cationic amino acid transporter, C-terminal / C-terminus of AA_permease / Amino acid/polyamine transporter I / Amino acid permease
Similarity search - Domain/homology
Lauryl Maltose Neopentyl Glycol / CHOLESTEROL / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / L-ornithine / Cationic amino acid transporter 4, vacuolar
Similarity search - Component
Biological speciessynthetic construct (others)
Arabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsKolokouris, D. / 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 16, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 30, 2026Provider: repository / Type: Initial release
Revision 1.0Sep 30, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: SybB5
A: Cationic amino acid transporter 4, vacuolar
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,6198
Polymers76,1882
Non-polymers3,4316
Water362
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

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Protein , 2 types, 2 molecules BA

#1: Protein SybB5


Mass: 12508.005 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: 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

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Non-polymers , 5 types, 8 molecules

#3: Chemical ChemComp-ORN / L-ornithine


Type: L-peptide linking / Mass: 132.161 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H12N2O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H88O22 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Complex of Arabidopsis thaliana cationic amino acid transporter 4 with a synthetic nanobody bound to L-ornithine, cholesterol and LMNG
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5460
Buffer solutionpH: 7.5 / Details: 20 mM Tris-HCl, 150 mM NaCl, 0.003% w/v LMNG
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was homogeneously monodisperse.
Specimen supportGrid material: COPPER / 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: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2PHENIX1.21_5207model refinement
12cryoSPARC4.7.1classification
13cryoSPARC4.7.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76191 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingPDB-ID: 9HJK

9hjk


Accession code: 9HJK / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.85 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00245081
ELECTRON MICROSCOPYf_angle_d0.55816924
ELECTRON MICROSCOPYf_chiral_restr0.0404822
ELECTRON MICROSCOPYf_plane_restr0.0038826
ELECTRON MICROSCOPYf_dihedral_angle_d12.71561031

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