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

Basic information

• Entry: Database: EMDB / ID: EMD-4386
• Title: cryo-EM structure of the human neutral amino acid transporter ASCT2

Sample

• Complex: human ASCT2 SLC1A5
  • Protein or peptide: Neutral amino acid transporter B(0)
• Ligand: GLUTAMINE

Function / homology

Function:

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

Domain/homology:

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.

Component:

Neutral amino acid transporter B(0)

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.85 Å
• Authors: Garaeva AA / Oostergetel GT / Gati C / Guskov A / Paulino C / Slotboom DJ

Funding support

Netherlands, 2 items

Organization	Grant number	Country
European Commission	MSCI 749732	Netherlands
European Commission	ERC 282083	Netherlands

• Citation: Journal: 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 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.

History

Deposition	Apr 19, 2018	-
Header (metadata) release	May 23, 2018	-
Map release	Jun 13, 2018	-
Update	Dec 11, 2019	-
Current status	Dec 11, 2019	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_4386.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.012 Å

Density

Contour Level	By AUTHOR: 0.036 / Movie #1: 0.036
Minimum - Maximum	-0.12015862 - 0.19263914
Average (Standard dev.)	0.00003537024 (±0.0069210045)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 240 240 240 Spacing 240 240 240
Cell	A=B=C: 242.87999 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.012	1.012	1.012
M x/y/z	240	240	240
origin x/y/z	0.000	0.000	0.000
length x/y/z	242.880	242.880	242.880
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	240	240	240
D min/max/mean	-0.120	0.193	0.000

Supplemental data

Mask #1

• File: emd_4386_msk_1.map

Additional map: #1

• File: emd_4386_additional.map

Half map: half-map 1 used for post processing step and...

• File: emd_4386_half_map_1.map
• Annotation: half-map 1 used for post processing step and FSC resolution calculation.

Half map: half-map 2 used for post processing step and...

• File: emd_4386_half_map_2.map
• Annotation: half-map 2 used for post processing step and FSC resolution calculation.

Sample components

Entire : human ASCT2 SLC1A5

• Entire: Name: human ASCT2 SLC1A5

Components

• Complex: human ASCT2 SLC1A5
  • Protein or peptide: Neutral amino acid transporter B(0)
• Ligand: GLUTAMINE

Supramolecule #1: human ASCT2 SLC1A5

• Supramolecule: Name: human ASCT2 SLC1A5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: human ASCT2 SLC1A5
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Komagataella pastoris (fungus)
• Molecular weight: Experimental: 172 KDa

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

• Macromolecule: Name: 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 weight: Theoretical: 56.638902 KDa
• Recombinant expression: Organism: Komagataella pastoris (fungus)

Sequence

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

Macromolecule #2: GLUTAMINE

• Macromolecule: Name: GLUTAMINE / type: ligand / ID: 2 / Number of copies: 3 / Formula: GLN
• Molecular weight: Theoretical: 146.144 Da

Chemical component information

ChemComp-GLN:
GLUTAMINE / Glutamine

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 2.5 mg/mL
• Buffer: pH: 7 ; Details: 20mM Tris-HCl pH 7.4 300mM NaCl 1mM L-glutamine 0.05% DDM 0.005% CHS
• Grid: Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK II

Electron microscopy

• Microscope: FEI TALOS ARCTICA
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Calibrated defocus max: 0.0025 µm / Calibrated defocus min: 0.0004 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.0025 µm / Nominal defocus min: 0.0004 µm / Nominal magnification: 49407
• Specialist optics: Energy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Temperature: Min: 70.0 K / Max: 90.0 K
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-60 / Average exposure time: 9.0 sec. / Average electron dose: 0.87 e/Ų; Details: 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
• Experimental equipment: Model: Talos Arctica / Image courtesy: FEI Company

Image processing

• CTF correction: Software - Name: CTFFIND (ver. 4.1)

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

3kbc

• Initial angle assignment: Type: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
• Final angle assignment: Type: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
• Final reconstruction: Applied symmetry - Point group: C₃ (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1); 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.; Number images used: 184080

Atomic model buiding 1

• Refinement: Space: REAL / Protocol: AB INITIO MODEL

Output model

PDB-6gct:
cryo-EM structure of the human neutral amino acid transporter ASCT2