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- EMDB-10016: Inward-open structure of the ASCT2 (SLC1A5) mutant C467R in prese... -

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Basic information

Entry
Database: EMDB / ID: EMD-10016
TitleInward-open structure of the ASCT2 (SLC1A5) mutant C467R in presence of TBOA
Map dataASCT2-C467R in the presence of TBOA at 3.6 %u212B resolution sharpened at -204 %u212B^2
Sample
  • Complex: C467R mutant of ASCT2 (Alanine Serine Cysteine Transporter 2, SLC1A)
    • Protein or peptide: Neutral amino acid transporter B(0)
Keywordssolute carrier family 1 (SLC1A) one-gate elevator mechanism neutral amino acid exchange membrane protein cryo-EM / MEMBRANE PROTEIN
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / L-glutamine transmembrane transporter activity / glutamine transport / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity ...glutamine secretion / L-glutamine import across plasma membrane / L-glutamine transmembrane transporter activity / glutamine transport / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / symporter activity / Amino acid transport across the plasma membrane / amino acid transmembrane transporter activity / antiporter activity / RHOJ GTPase cycle / protein homotrimerization / RHOQ GTPase cycle / amino acid transport / RHOH GTPase cycle / RAC3 GTPase cycle / transport across blood-brain barrier / RAC1 GTPase cycle / basal plasma membrane / erythrocyte differentiation / centriolar satellite / melanosome / signaling receptor activity / virus receptor activity / ciliary basal body / extracellular exosome / metal ion binding / membrane / 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: 3.61 Å
AuthorsGaraeva AA / Guskov A
Funding support Netherlands, 5 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research723.014.002 Netherlands
Netherlands Organisation for Scientific Research865.11.001 Netherlands
European Research Council282083 Netherlands
Netherlands Organisation for Scientific Research722.017.001 Netherlands
Netherlands Organisation for Scientific Research740.018.016 Netherlands
CitationJournal: Nat Commun / Year: 2019
Title: A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2.
Authors: Alisa A Garaeva / Albert Guskov / Dirk J Slotboom / Cristina Paulino /
Abstract: The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type ...The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.
History
DepositionJun 3, 2019-
Header (metadata) releaseJun 12, 2019-
Map releaseAug 7, 2019-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0607
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0607
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rvx
  • Surface level: 0.0607
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10016.png

Downloads & links

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Map

FileDownload / File: emd_10016.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationASCT2-C467R in the presence of TBOA at 3.6 %u212B resolution sharpened at -204 %u212B^2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 200 pix.
= 202.4 Å		1.01 Å/pix.
x 200 pix.
= 202.4 Å		1.01 Å/pix.
x 200 pix.
= 202.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.012 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0607 / Movie #1: 0.0607
Minimum - Maximum-0.3030893 - 0.41591617
Average (Standard dev.)0.0011954098 (±0.011214978)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 202.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z202.400202.400202.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ10810112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.3030.4160.001

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Supplemental data

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Mask #1

Fileemd_10016_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_10016_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_10016_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : C467R mutant of ASCT2 (Alanine Serine Cysteine Transporter 2, SLC1A)

EntireName: C467R mutant of ASCT2 (Alanine Serine Cysteine Transporter 2, SLC1A)
Components
  • Complex: C467R mutant of ASCT2 (Alanine Serine Cysteine Transporter 2, SLC1A)
    • Protein or peptide: Neutral amino acid transporter B(0)

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Supramolecule #1: C467R mutant of ASCT2 (Alanine Serine Cysteine Transporter 2, SLC1A)

SupramoleculeName: C467R mutant of ASCT2 (Alanine Serine Cysteine Transporter 2, SLC1A)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 172 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: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.521848 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 SRTVLNVEGD ALGAG LLQN YVDRTESRST EPELIQVKSE LPLDPLPVPT EEGNPLLKHY RGPAGDATVA SEKESVMHHH HHH

UniProtKB: Neutral amino acid transporter B(0)

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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 mg/mL
BufferpH: 7.4
Details: 20 mM Tris pH 7.4, 300 mM NaCl, 0.05% DDM, 0.005% CHS
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
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
TemperatureMin: 90.0 K / Max: 105.0 K
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 - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 6 / Number real images: 7327 / Average exposure time: 9.0 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 49407
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: 1109646
Startup modelType of model: EMDB MAP
EMDB ID:

4386

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta-2) / Number images used: 223354
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0-beta-2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0-beta-2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6gct


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

PDB-6rvx:
Inward-open structure of the ASCT2 (SLC1A5) mutant C467R in presence of TBOA

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