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- EMDB-30341: Consensus mutated xCT-CD98hc complex -

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Basic information

Entry
Database: EMDB / ID: EMD-30341
TitleConsensus mutated xCT-CD98hc complex
Map dataMain map
Sample
  • Complex: Consensus mutated xCT-CD98hc complex
    • Protein or peptide: 4F2 cell-surface antigen heavy chain
    • Protein or peptide: Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc- System
Function / homology
Function and homology information


cystine:glutamate antiporter activity / regulation of neutrophil apoptotic process / L-kynurenine transmembrane transport / L-kynurenine transmembrane transporter activity / regulation of cysteine metabolic process / regulation of glutathione biosynthetic process / regulation of glutamate metabolic process / regulation of melanin biosynthetic process / regulation of AMPA glutamate receptor clustering / L-cystine transport ...cystine:glutamate antiporter activity / regulation of neutrophil apoptotic process / L-kynurenine transmembrane transport / L-kynurenine transmembrane transporter activity / regulation of cysteine metabolic process / regulation of glutathione biosynthetic process / regulation of glutamate metabolic process / regulation of melanin biosynthetic process / regulation of AMPA glutamate receptor clustering / L-cystine transport / negative regulation of ferroptosis / dipeptide import across plasma membrane / neutral L-amino acid secondary active transmembrane transporter activity / Defective SLC7A7 causes lysinuric protein intolerance (LPI) / methionine transport / tyrosine transport / L-histidine transport / apical pole of neuron / aromatic amino acid transmembrane transporter activity / amino acid transport complex / L-amino acid transmembrane transporter activity / : / L-leucine import across plasma membrane / L-alanine transmembrane transporter activity / isoleucine transport / L-alanine import across plasma membrane / regulation of protein transport / phenylalanine transport / valine transport / L-leucine transmembrane transporter activity / amino acid transmembrane transport / calcium:sodium antiporter activity / L-leucine transport / thyroid hormone transport / proline transport / L-glutamate transmembrane transport / glutathione transmembrane transport / regulation of cellular response to oxidative stress / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / ventricular system development / intracellular glutamate homeostasis / lens fiber cell differentiation / L-glutamate import across plasma membrane / Tryptophan catabolism / striatum development / astrocyte projection / exogenous protein binding / anchoring junction / limb development / Basigin interactions / response to redox state / NFE2L2 regulating anti-oxidant/detoxification enzymes / microvillus membrane / amino acid transport / adult behavior / regulation of synapse organization / lung alveolus development / response to exogenous dsRNA / tryptophan transport / glutathione metabolic process / basal plasma membrane / brush border membrane / response to nicotine / visual learning / modulation of chemical synaptic transmission / response to organic cyclic compound / response to toxic substance / platelet aggregation / calcium ion transport / double-stranded RNA binding / melanosome / apical part of cell / virus receptor activity / regulation of cell population proliferation / cellular response to oxidative stress / basolateral plasma membrane / carbohydrate metabolic process / cadherin binding / symbiont entry into host cell / apical plasma membrane / protein heterodimerization activity / lysosomal membrane / synapse / cell surface / protein homodimerization activity / RNA binding / extracellular exosome / nucleoplasm / membrane / plasma membrane
Similarity search - Function
L-type amino acid transporter / Solute carrier family 3 member 2, N-terminal domain / 4F2 cell-surface antigen heavy chain / Solute carrier family 3 member 2 N-terminus / Amino acid/polyamine transporter I / Amino acid permease / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Amino acid transporter heavy chain SLC3A2 / Cystine/glutamate transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsOda K / Lee Y / Takemoto M / Yamashita K / Nishizawa T / Nureki O
CitationJournal: Protein Sci / Year: 2020
Title: Consensus mutagenesis approach improves the thermal stability of system x transporter, xCT, and enables cryo-EM analyses.
Authors: Kazumasa Oda / Yongchan Lee / Pattama Wiriyasermkul / Yoko Tanaka / Mizuki Takemoto / Keitaro Yamashita / Shushi Nagamori / Tomohiro Nishizawa / Osamu Nureki /
Abstract: System x is an amino acid antiporter that imports L-cystine into cells and exports intracellular L-glutamate, at a 1:1 ratio. As L-cystine is an essential precursor for glutathione synthesis, system ...System x is an amino acid antiporter that imports L-cystine into cells and exports intracellular L-glutamate, at a 1:1 ratio. As L-cystine is an essential precursor for glutathione synthesis, system x supports tumor cell growth through glutathione-based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System x consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L-type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single-particle analyses of the xCT-CD98hc complex. As wild-type xCT-CD98hc displayed poor stability and could not be purified to homogeneity, we applied a consensus mutagenesis approach to xCT. The consensus mutated construct exhibited increased stability as compared to the wild-type, and enabled the cryoelectron microscopy (cryo-EM) map to be obtained at 6.2 Å resolution by single-particle analysis. The cryo-EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our consensus-based strategy could represent a good choice toward solving unstable protein structures.
History
DepositionJun 17, 2020-
Header (metadata) releaseDec 9, 2020-
Map releaseDec 9, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.027
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  • Surface view with fitted model
  • Atomic models: PDB-7ccs
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ccs
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30341.png

Downloads & links

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Map

FileDownload / File: emd_30341.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Voxel sizeX=Y=Z: 1.47556 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.027
Minimum - Maximum-0.047360767 - 0.09613854
Average (Standard dev.)0.000430362 (±0.0037140287)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 265.6008 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.47556111111111.47556111111111.4755611111111
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z265.601265.601265.601
α/β/γ90.00090.00090.000
start NX/NY/NZ107107101
NX/NY/NZ267267267
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0470.0960.000

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Supplemental data

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Mask #1

Fileemd_30341_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half2

Fileemd_30341_half_map_1.map
AnnotationHalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half1

Fileemd_30341_half_map_2.map
AnnotationHalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Consensus mutated xCT-CD98hc complex

EntireName: Consensus mutated xCT-CD98hc complex
Components
  • Complex: Consensus mutated xCT-CD98hc complex
    • Protein or peptide: 4F2 cell-surface antigen heavy chain
    • Protein or peptide: Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc- System

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Supramolecule #1: Consensus mutated xCT-CD98hc complex

SupramoleculeName: Consensus mutated xCT-CD98hc complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 120 KDa

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Macromolecule #1: 4F2 cell-surface antigen heavy chain

MacromoleculeName: 4F2 cell-surface antigen heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.069625 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSELQPPEAS IAVVSIPRQL PGSHSEAGVQ GLSAGDDSEL GSHCVAQTGL ELLASGDPLP SASQNAEMIE TGSDCVTQAG LQLLASSDP PALASKNAEV TGTMSQDTEV DMKEVELNEL EPEKQPMNAA SGAAMSLAGA EKNGLVKIKV AEDEAEAAAA A KFTGLSKE ...String:
GSELQPPEAS IAVVSIPRQL PGSHSEAGVQ GLSAGDDSEL GSHCVAQTGL ELLASGDPLP SASQNAEMIE TGSDCVTQAG LQLLASSDP PALASKNAEV TGTMSQDTEV DMKEVELNEL EPEKQPMNAA SGAAMSLAGA EKNGLVKIKV AEDEAEAAAA A KFTGLSKE ELLKVAGSPG WVRTRWALLL LFWLGWLGML AGAVVIIVRA PRCRELPAQK WWHTGALYRI GDLQAFQGHG AG NLAGLKG RLDYLSSLKV KGLVLGPIHK NQKDDVAQTD LLQIDPNFGS KEDFDSLLQS AKKKSIRVIL DLTPNYRGEN SWF STQVDT VATKVKDALE FWLQAGVDGF QVRDIENLKD ASSFLAEWQN ITKGFSEDRL LIAGTNSSDL QQILSLLESN KDLL LTSSY LSDSGSTGEH TKSLVTQYLN ATGNRWCSWS LSQARLLTSF LPAQLLRLYQ LMLFTLPGTP VFSYGDEIGL DAAAL PGQP MEAPVMLWDE SSFPDIPGAV SANMTVKGQS EDPGSLLSLF RRLSDQRSKE RSLLHGDFHA FSAGPGLFSY IRHWDQ NER FLVVLNFGDV GLSAGLQASD LPASASLPAK ADLLLSTQPG REEGSPLELE RLKLEPHEGL LLRFPYAA

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Macromolecule #2: Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc-...

MacromoleculeName: Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc- System
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.933852 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVRKPVVSTI SKGGYLQGNV NGRLPSLGSK EPPGQEKVQL KREITLLDGV SLIVGTIIGA GIFVSPKGVL KNTGSVGLSL VIWAVCGVL SLFGALCYAE LGTTIPKSGG AYLYILETFG PLPAFLRGWN ELLIIRPAST AVISLAFGNY ILEPFFPTCE P PELAIKLL ...String:
MVRKPVVSTI SKGGYLQGNV NGRLPSLGSK EPPGQEKVQL KREITLLDGV SLIVGTIIGA GIFVSPKGVL KNTGSVGLSL VIWAVCGVL SLFGALCYAE LGTTIPKSGG AYLYILETFG PLPAFLRGWN ELLIIRPAST AVISLAFGNY ILEPFFPTCE P PELAIKLL AAVGILLLTV LNSLSVKWSA RVQDFFTAAK LLALLIIIVP GVVQLIKGQT QNFKDAFEGS DPSIGGLPLA FY SGLYAYV GWDYLNFVTE EVKNPEKNIP LAIVISMPIV TVAYVLTNVA YFTTLSPEEL LLSNAVAVTF GERLLGNFSW AVP IFVALS CFGSLNGSLF AMSRLFYVAA REGHLPKILS MIHVRRHTPL PALIVSGPLT AIMLFLGDLF SLINFMSFGT WLFY GLVVA GLIYLRYKKP DLHRPIKVPL FIPILFLLTC LFLVAVSLYS DPVNCGIGFV IILTGVPVYF LFVYWDKKPK WFRRI SEKI TRHLQLLLEV VPEEDKLDYK DDDDK

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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

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.76 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF
Details: This pdb entry contains an issue about peptide linkage (bond outlier). Residues GLU A 213 and LEU A 214 that are next to each other are not properly linked: distance between C and N is 3.35. ...Details: This pdb entry contains an issue about peptide linkage (bond outlier). Residues GLU A 213 and LEU A 214 that are next to each other are not properly linked: distance between C and N is 3.35. This was introduced upon the fitting of the homology model into the low resolution cryo-EM map by using Rosetta. The model has not undergone any further structure refinement.
Number images used: 86395
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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