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- EMDB-55065: Cryo-EM structure of the Arabidopsis thaliana CAT4 transporter in... -

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Basic information

Entry
Database: EMDB / ID: EMD-55065
TitleCryo-EM structure of the Arabidopsis thaliana CAT4 transporter in the outward-open L-ornithine bound state
Map dataNone
Sample
  • Complex: Complex of Arabidopsis thaliana cationic amino acid transporter 4 with a synthetic nanobody bound to L-ornithine, cholesterol and LMNG
    • Protein or peptide: SybB5
    • Protein or peptide: Cationic amino acid transporter 4, vacuolar
  • Ligand: L-ornithine
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: CHOLESTEROL
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: water
Keywordsmembrane transporter / cationic amino acid / TRANSPORT 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsKolokouris D / Newstead S
Funding support United Kingdom, 1 items
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, 2025-
Header (metadata) releaseSep 30, 2026-
Map releaseSep 30, 2026-
UpdateSep 30, 2026-
Current statusSep 30, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_55065.png

Downloads & links

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Map

FileDownload / File: emd_55065.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 288 pix.
= 240.48 Å		0.84 Å/pix.
x 288 pix.
= 240.48 Å		0.84 Å/pix.
x 288 pix.
= 240.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.075
Minimum - Maximum-0.5532741 - 0.67372584
Average (Standard dev.)0.00006252805 (±0.013889313)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 240.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_55065_msk_1.map
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Additional map: None

Fileemd_55065_additional_1.map
AnnotationNone
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Half map: None

Fileemd_55065_half_map_1.map
AnnotationNone
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Half map: #1

Fileemd_55065_half_map_2.map
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Sample components

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Entire : Complex of Arabidopsis thaliana cationic amino acid transporter 4...

EntireName: Complex of Arabidopsis thaliana cationic amino acid transporter 4 with a synthetic nanobody bound to L-ornithine, cholesterol and LMNG
Components
  • Complex: Complex of Arabidopsis thaliana cationic amino acid transporter 4 with a synthetic nanobody bound to L-ornithine, cholesterol and LMNG
    • Protein or peptide: SybB5
    • Protein or peptide: Cationic amino acid transporter 4, vacuolar
  • Ligand: L-ornithine
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: CHOLESTEROL
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: water

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Supramolecule #1: Complex of Arabidopsis thaliana cationic amino acid transporter 4...

SupramoleculeName: Complex of Arabidopsis thaliana cationic amino acid transporter 4 with a synthetic nanobody bound to L-ornithine, cholesterol and LMNG
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: SybB5

MacromoleculeName: SybB5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.508005 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGFPVN MYWMHWYRQA PGKEREWVAA IQSYGQWTAY ADSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCAVG VGGYYLGQGT QVTVS

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Macromolecule #2: Cationic amino acid transporter 4, vacuolar

MacromoleculeName: Cationic amino acid transporter 4, vacuolar / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 63.680152 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MNSLVRRKQV DSVHLIKNDG PHQLAKKLSA VDLVAIGVGT TIGAGVYILV GTVAREHTGP ALAVSFFIAG VAAALSACCY AELASRCPS AGSAYHYAYI CLGEGIAWLV GWALVLDYTI GGSAIARGIT PNLASFFGGL DNLPVFLARQ TIPGVGIVVD P CAALLIMI ...String:
MNSLVRRKQV DSVHLIKNDG PHQLAKKLSA VDLVAIGVGT TIGAGVYILV GTVAREHTGP ALAVSFFIAG VAAALSACCY AELASRCPS AGSAYHYAYI CLGEGIAWLV GWALVLDYTI GGSAIARGIT PNLASFFGGL DNLPVFLARQ TIPGVGIVVD P CAALLIMI VTILLCFGIK ESSTVQAIVT SVNVCTLVFI IVVGGYLACK TGWVGYDLPS GYFPFGLNGI LAGSAVVFFS YI GFDTVTS TAEEVKNPQR DLPLGIGIAL LICCILYMLL SVVIVGLVPY YSLNPDTPIS SAFGDSGMQW AAYILTTGAI TAL CASLLG SLLAQPRIFM AMARDGLLPA FFSEISPRTQ VPVKSTIAIG VLAAALAFFM DVAQLSEMVS VGTLMAFTAV AVCV LVLRY VPPDGVPLSS SSQTLSDTDE SRAETENFLV DAIESSDSPL LGNETARDEK YFGKRRKIAA WSIALVCIGV LGLAS AASA ERLPSFPRFT ICGVSAVILL GSLITLGYID EDEERHNFGH KGGFLCPFVP YLPVLCILIN TYLIINIGAG TWIRVL IWL LIGSMIYIFY GRSHSLLNNA VYVPTMTCTR KTTDHLA

UniProtKB: Cationic amino acid transporter 4, vacuolar

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Macromolecule #3: L-ornithine

MacromoleculeName: L-ornithine / type: ligand / ID: 3 / Number of copies: 1 / Formula: ORN
Molecular weightTheoretical: 132.161 Da
Chemical component information

ChemComp-ORN:
L-ornithine

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Macromolecule #4: Lauryl Maltose Neopentyl Glycol

MacromoleculeName: Lauryl Maltose Neopentyl Glycol / type: ligand / ID: 4 / Number of copies: 1 / Formula: AV0
Molecular weightTheoretical: 1.005188 KDa
Chemical component information

ChemComp-AV0:
Lauryl Maltose Neopentyl Glycol

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 2 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #6: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

MacromoleculeName: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / type: ligand / ID: 6 / Number of copies: 2 / Formula: LBN
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-LBN:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / phospholipid*YM

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration5.0 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris-HCl, 150 mM NaCl, 0.003% w/v LMNG
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was homogeneously monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 76191
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9hjk


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation coefficient
Output model

PDB-9sp8:
Cryo-EM structure of the Arabidopsis thaliana CAT4 transporter in the outward-open L-ornithine bound state

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