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- EMDB-18628: ASCT2 protomer in lipid nanodiscs under low Na+ concentration in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-18628
TitleASCT2 protomer in lipid nanodiscs under low Na+ concentration in the intermediate outward-facing state (iOFS-up)
Map dataSharpened map of ASCT2 protomer in 2N2 under low Na concentration in iOFS-up with Phenix
Sample
  • Complex: ASCT2 in MSP2N2 lipid nanodiscs in the presence of K+ ions
    • Protein or peptide: Neutral amino acid transporter B(0)
  • Ligand: SODIUM ION
Keywordsneutral amino acid exchanger amino acid transport system elevator transporter glutamine transport / TRANSPORT PROTEIN
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / glutamine transport / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / L-aspartate transmembrane transporter activity / Amino acid transport across the plasma membrane ...glutamine secretion / L-glutamine import across plasma membrane / glutamine transport / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / L-aspartate transmembrane transporter activity / Amino acid transport across the plasma membrane / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / symporter activity / antiporter activity / amino acid transport / RHOJ GTPase cycle / RHOQ GTPase cycle / protein homotrimerization / RHOH GTPase cycle / transport across blood-brain barrier / RAC3 GTPase cycle / RAC1 GTPase cycle / basal plasma membrane / erythrocyte differentiation / melanosome / signaling receptor activity / virus receptor activity / extracellular exosome / membrane / metal ion binding / plasma membrane
Similarity search - Function
: / Sodium:dicarboxylate symporter family signature 2. / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1.
Similarity search - Domain/homology
Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsBorowska A / Rheinberger J / Paulino C / Slotboom DJ
Funding support Netherlands, 2 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)40.018.016 Netherlands
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of the obligatory exchange mode of human neutral amino acid transporter ASCT2.
Authors: Anna M Borowska / Maria Gabriella Chiariello / Alisa A Garaeva / Jan Rheinberger / Siewert J Marrink / Cristina Paulino / Dirk J Slotboom /
Abstract: ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that ...ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that concentrate glutamate in the cytosol. The mechanism that makes ASCT2 an exchanger rather than a concentrator remains enigmatic. Here, we employ cryo-electron microscopy and molecular dynamics simulations to elucidate the structural basis of the exchange mechanism of ASCT2. We establish that ASCT2 binds three Na ions per transported substrate and visits a state that likely acts as checkpoint in preventing Na ion leakage, both features shared with EAATs. However, in contrast to EAATs, ASCT2 retains one Na ion even under Na-depleted conditions. We demonstrate that ASCT2 cannot undergo the structural transition in TM7 that is essential for the concentrative transport cycle of EAATs. This structural rigidity and the high-affinity Na binding site effectively confine ASCT2 to an exchange mode.
History
DepositionOct 9, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18628.png

Downloads & links

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Map

FileDownload / File: emd_18628.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of ASCT2 protomer in 2N2 under low Na concentration in iOFS-up with Phenix
Voxel sizeX=Y=Z: 1.022 Å
Density
Contour LevelBy AUTHOR: 4.4
Minimum - Maximum-21.360676000000002 - 32.820520000000002
Average (Standard dev.)-0.00014792154 (±0.5940769)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 261.632 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18628_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ASCT2 in MSP2N2 lipid nanodiscs in the presence of K+ ions

EntireName: ASCT2 in MSP2N2 lipid nanodiscs in the presence of K+ ions
Components
  • Complex: ASCT2 in MSP2N2 lipid nanodiscs in the presence of K+ ions
    • Protein or peptide: Neutral amino acid transporter B(0)
  • Ligand: SODIUM ION

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Supramolecule #1: ASCT2 in MSP2N2 lipid nanodiscs in the presence of K+ ions

SupramoleculeName: ASCT2 in MSP2N2 lipid nanodiscs in the presence of K+ ions
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 53.4 KDa

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Macromolecule #1: Neutral amino acid transporter B(0)

MacromoleculeName: Neutral amino acid transporter B(0) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.467793 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MVADPPRDSK GLAAAEPTAN GGLALASIED QGAAAGGYCG SRDQVRRCLR ANLLVLLTVV AVVAGVALGL GVSGAGGALA LGPERLSAF VFPGELLLRL LRMIILPLVV CSLIGGAASL DPGALGRLGA WALLFFLVTT LLASALGVGL ALALQPGAAS A AINASVGA ...String:
MVADPPRDSK GLAAAEPTAN GGLALASIED QGAAAGGYCG SRDQVRRCLR ANLLVLLTVV AVVAGVALGL GVSGAGGALA LGPERLSAF VFPGELLLRL LRMIILPLVV CSLIGGAASL DPGALGRLGA WALLFFLVTT LLASALGVGL ALALQPGAAS A AINASVGA AGSAENAPSK EVLDSFLDLA RNIFPSNLVS AAFRSYSTTY EERNITGTRV KVPVGQEVEG MNILGLVVFA IV FGVALRK LGPEGELLIR FFNSFNEATM VLVSWIMWYA PVGIMFLVAG KIVEMEDVGL LFARLGKYIL CCLLGHAIHG LLV LPLIYF LFTRKNPYRF LWGIVTPLAT AFGTSSSSAT LPLMMKCVEE NNGVAKHISR FILPIGATVN MDGAALFQCV AAVF IAQLS QQSLDFVKII TILVTATASS VGAAGIPAGG VLTLAIILEA VNLPVDHISL ILAVDWLVDR SCTVLNVEGD ALGAG LLQN YVDRTESRST EPELIQVKSE LPLDPLPVPT EEGNPLLKHY RGPAGDATVA SEKESVMHHH HHH

UniProtKB: Neutral amino acid transporter B(0)

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Macromolecule #2: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 2 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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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

Concentration1.6 mg/mL
BufferpH: 7.4 / Details: 20 mM Tris pH 7.4, 200 mM KCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-60 / Number grids imaged: 2 / Number real images: 4145 / Average exposure time: 9.0 sec. / Average electron dose: 50.9 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 48924 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3256089
Startup modelType of model: INSILICO MODEL / In silico model: In silico model generated with cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0)
Details: The number of particles is the number of protomers from C3 expanded particles
Number images used: 132084
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mpb


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8qrw:
ASCT2 protomer in lipid nanodiscs under low Na+ concentration in the intermediate outward-facing state (iOFS-up)

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