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- EMDB-9187: Cryo-EM structure of the human neutral amino acid transporter ASCT2 -

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Basic information

Entry
Database: EMDB / ID: EMD-9187
TitleCryo-EM structure of the human neutral amino acid transporter ASCT2
Map dataNeutral amino acid transporterAmino acid transporter
Sample
  • Complex: Ternary complex of SLC1A5
    • Protein or peptide: Neutral amino acid transporter B(0)
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / L-serine transmembrane transporter activity / glutamine transport / L-glutamine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity ...glutamine secretion / L-glutamine import across plasma membrane / L-serine transmembrane transporter activity / glutamine transport / L-glutamine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / symporter activity / amino acid transport / antiporter activity / 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 / virus receptor activity / signaling 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: 3.54 Å
AuthorsYu X / Han S
CitationJournal: Elife / Year: 2019
Title: Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation.
Authors: Xiaodi Yu / Olga Plotnikova / Paul D Bonin / Timothy A Subashi / Thomas J McLellan / Darren Dumlao / Ye Che / Yin Yao Dong / Elisabeth P Carpenter / Graham M West / Xiayang Qiu / Jeffrey S Culp / Seungil Han /
Abstract: Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned ...Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.
History
DepositionOct 5, 2018-
Header (metadata) releaseOct 31, 2018-
Map releaseNov 6, 2019-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mp6
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9187.png

Downloads & links

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Map

FileDownload / File: emd_9187.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNeutral amino acid transporter
Voxel sizeX=Y=Z: 1.086 Å
Density
Contour LevelBy AUTHOR: 2 / Movie #1: 2
Minimum - Maximum-7.2814302 - 12.210781
Average (Standard dev.)0.025915502 (±0.37348923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 243.26399 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0861.0861.086
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z243.264243.264243.264
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-7.28112.2110.026

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Supplemental data

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Mask #1

Fileemd_9187_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: human neutral amino acid transporter, half map #1

Fileemd_9187_half_map_1.map
Annotationhuman neutral amino acid transporter, half map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: human neutral amino acid transporter, half map #2

Fileemd_9187_half_map_2.map
Annotationhuman neutral amino acid transporter, half map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of SLC1A5

EntireName: Ternary complex of SLC1A5
Components
  • Complex: Ternary complex of SLC1A5
    • Protein or peptide: Neutral amino acid transporter B(0)
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Ternary complex of SLC1A5

SupramoleculeName: Ternary complex of SLC1A5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 170 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: 56.638902 KDa
Recombinant expressionOrganism: Homo sapiens (human)
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 SEKESVM

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration2 mg/mL
BufferpH: 7.5
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.06 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 165067

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