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- EMDB-4386: cryo-EM structure of the human neutral amino acid transporter ASCT2 -

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Basic information

Entry
Database: EMDB / ID: EMD-4386
Titlecryo-EM structure of the human neutral amino acid transporter ASCT2
Map data
Samplehuman ASCT2 SLC1A5:
Neutral amino acid transporter B(0) / ligand
Function / homology
Function and homology information


Amino acid transport across the plasma membrane / L-glutamine import across plasma membrane / glutamine transport / glutamine secretion / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / amino acid transmembrane transporter activity / neutral amino acid transport / neutral amino acid transmembrane transporter activity / symporter activity ...Amino acid transport across the plasma membrane / L-glutamine import across plasma membrane / glutamine transport / glutamine secretion / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / amino acid transmembrane transporter activity / neutral amino acid transport / neutral amino acid transmembrane transporter activity / symporter activity / amino acid transport / protein homotrimerization / virus receptor activity / melanosome / signaling receptor activity / integral component of plasma membrane / extracellular exosome / membrane / integral component of membrane / plasma membrane / metal ion binding
Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter family signature 2. / Sodium:dicarboxylate symporter family signature 1. / Sodium:dicarboxylate symporter family
Neutral amino acid transporter B(0)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsGaraeva AA / Oostergetel GT / Gati C / Guskov A / Paulino C / Slotboom DJ
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2018
Title: Cryo-EM structure of the human neutral amino acid transporter ASCT2.
Authors: Alisa A Garaeva / Gert T Oostergetel / Cornelius Gati / Albert Guskov / Cristina Paulino / Dirk J Slotboom /
Abstract: Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for ...Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses. Relative to structures of other SLC1 members, ASCT2 is in the most extreme inward-oriented state, with the transport domain largely detached from the central scaffold domain on the cytoplasmic side. This domain detachment may be required for substrate binding and release on the intracellular side of the membrane.
Validation ReportPDB-ID: 6gct

SummaryFull reportAbout validation report
DateDeposition: Apr 19, 2018 / Header (metadata) release: May 23, 2018 / Map release: Jun 13, 2018 / Update: Jun 20, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6gct
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4386.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 240 pix.
= 242.88 Å
1.01 Å/pix.
x 240 pix.
= 242.88 Å
1.01 Å/pix.
x 240 pix.
= 242.88 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
Contour LevelBy AUTHOR: 0.036 / Movie #1: 0.036
Minimum - Maximum-0.12015862 - 0.19263914
Average (Standard dev.)0.000035370238 (±0.0069210045)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 242.87999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z242.880242.880242.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1200.1930.000

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Supplemental data

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Mask #1

Fileemd_4386_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire human ASCT2 SLC1A5

EntireName: human ASCT2 SLC1A5 / Details: human ASCT2 SLC1A5 / Number of components: 3

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Component #1: protein, human ASCT2 SLC1A5

ProteinName: human ASCT2 SLC1A5 / Details: human ASCT2 SLC1A5 / Recombinant expression: No
MassExperimental: 172 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Komagataella pastoris (fungus)

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Component #2: protein, Neutral amino acid transporter B(0)

ProteinName: Neutral amino acid transporter B(0) / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 56.638902 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Komagataella pastoris (fungus)

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Component #3: ligand, GLUTAMINE

LigandName: GLUTAMINE / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.146144 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/mL
Buffer solution: 20mM Tris-HCl pH 7.4 300mM NaCl 1mM L-glutamine 0.05% DDM 0.005% CHS
pH: 7
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temperature: 278 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.87 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 49407.0 X (nominal), 49407.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 0.4 - 2.5 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (70.0 - 90.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionDetails: Freshly purified protein was concentrated using Vivaspin concentrating devices with a molecular weight cutoff of 100kDa to 2-2.5 mg ml-1. 2.8 ul were applied on holey-carbon cryo-EM grids (Quantifoil Au R1.2-1.3, 200 and 300 mesh), which were prior glow-discharged at 5 mA for 20 s. Grids were blotted for 3-5 s in a Vitrobot (Mark 3, Thermo Fisher) at 20C temperature and 100% humidity, subsequently plunge-frozen in liquid ethane and stored in liquid nitrogen. Cryo-EM data were collected on a 200 keV Talos Arctica microscope (Thermo Fisher) using a post-column energy filter (Gatan) in zero-loss mode, using a 20 eV slit, a 100 um objective aperture, in an automated fashion using EPU software (Thermo Fisher) on a K2 summit detector (Gatan) in counting mode. Cryo-EM images were acquired at a pixel size of 1.012A (calibrated magnification of 49,407x), a defocus range from -0.4 to 2.5 um, an exposure time of 9 sec and a sub-frame exposure time of 150 ms (60 frames), and a total electron dose on the specimen level of about 52 electrons per A2. Best regions on the grid were screened with a self-written script to calculate the ice thickness and data quality was monitored on the fly using the software FOCUS

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 184080
3D reconstructionSoftware: RELION / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF
Details: A total of 6345 dose-fractionated cryo-EM images were recorded and subjected to motion-correction and dose-weighting of frames by MotionCor2. The CTF parameters were estimated on the movie frames by ctffind4.1. Bad images showing contamination, a defocus below or above 0.4 and -3um or a bad CTF estimation were discarded, resulting in 4863 images used for further analysis with the software package RELION2.1. About 3000 particles were picked manually to generate 2D references which where improved in several rounds of autopick. A low threshold was used during the final autopick step to ensure that no particles are missed yielding more than a million particles. Particles were extracted with a box size of 240 pixels, and initial classification steps were performed with three-fold binned data. False positives or bad particles were removed in first rounds of 2D classification, resulting in 628,015 particles that were further sorted in several rounds of 3D classification. A map generated from the GltPh structure (PDB ID 3KBC) was used as reference for the first round, and the best output class was used in subsequent jobs in an iterative way. The best 3D class, comprising 184,080 particles from 4859 images, was subjected to auto-refinement, yielding a map with a resolution of 4.26 A before masking and 3.91 A after masking. Particles were further polished in RELION version 2.1 and subjected to another round of 2D and 3D classification resulting in a final dataset of 133,437 particles. The final polished map had a resolution of 4.26 A before masking and 3.85 A after masking. The map was sharpened using an isotropic B-factor of -171 A2, for manual inspection a B-factor of -225 A2 was used. The approach of focused refinement, where the less-resolved detergent micelle was subtracted from the particle images, did not improve resolution. During 3D classification and auto-refinement jobs a C3-symmetry was imposed. To check for conformational heterogeneity of the data, where single protomers within the trimer might adopt a different conformation, 3D classifications with no symmetry imposed were performed at different stages of image processing. We further performed 3D classification on the individual protomers of a single transporter using symmetry expansion and signal subtraction. Both approaches showed no indication of the existence of a different conformation. Local resolution estimates were estimated by RELION. All resolutions were estimated using the 0.143 cut-off criterion with gold-standard Fourier shell correlation (FSC) between two independently refined half maps. During post-processing, the approach of high-resolution noise substitution was used to correct for convolution effects of real-space masking on the FSC curve.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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