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

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Basic information

Entry
Database: PDB / ID: 9hjk
TitleCryo-EM structure of the Arabidopsis thaliana CAT4 transporter in the outward-open apo state
Components
  • Cationic amino acid transporter 4, vacuolar
  • SybB5
KeywordsMEMBRANE PROTEIN / cationic amino acid transporter / APC fold / LeuT fold / cholesterol / outward-open state / cationic amino acids / SLC7
Function / homology
Function and homology information


plant-type vacuole membrane / plant-type vacuole / amino acid transport / transmembrane transporter activity
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 / Cationic amino acid transporter 4, vacuolar
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsKolokouris, D. / Kato, T. / Newstead, S.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011224/1 United Kingdom
Citation
Journal: To Be Published
Title: Cryo-EM structure of the Arabidopsis thaliana CAT4 transporter in the outward-open state
Authors: Kolokouris, D. / Newstead, S.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionNov 29, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 10, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)73,6084
Polymers72,2162
Non-polymers1,3922
Water181
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: 59390.160 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
#2: Protein SybB5


Mass: 12826.291 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)
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O / 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
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 and cholesterol
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #2, #1 / 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.: 3.4 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: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2RELIONparticle selection
3PHENIX1.21_5207model refinement
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11166793
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 642325 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingAccession code: AF-Q8W4K3-F1 / Source name: AlphaFold / Type: in silico model

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