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- EMDB-11726: Sub-nanometer cryo-EM density map of the human heterodimeric amin... -

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Basic information

Entry
Database: EMDB / ID: EMD-11726
TitleSub-nanometer cryo-EM density map of the human heterodimeric amino acid transporter 4F2hc-LAT2
Map data
Sample
  • Organelle or cellular component: 4F2hc-LAT2
    • Protein or peptide: hLAT2
    • Protein or peptide: h4F2hc
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsJeckelmann J-M / Fotiadis D
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science FoundationNational Centre of Competence in Research (NCCR) TransCure Switzerland
CitationJournal: Int J Mol Sci / Year: 2020
Title: Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.
Authors: Jean-Marc Jeckelmann / Dimitrios Fotiadis /
Abstract: Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a ...Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast , and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed.
History
DepositionSep 14, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateOct 14, 2020-
Current statusOct 14, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11726.png

Downloads & links

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Map

FileDownload / File: emd_11726.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.66 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum-0.94068015 - 2.2950344
Average (Standard dev.)0.00003898510 (±0.12522496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.661.661.66
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z298.800298.800298.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ161186271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.9412.2950.000

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Supplemental data

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Sample components

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Entire : 4F2hc-LAT2

EntireName: 4F2hc-LAT2
Components
  • Organelle or cellular component: 4F2hc-LAT2
    • Protein or peptide: hLAT2
    • Protein or peptide: h4F2hc

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Supramolecule #1: 4F2hc-LAT2

SupramoleculeName: 4F2hc-LAT2 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: heavy and light chain of the contently linked heterodimeric amino acid transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 KDa
Recombinant expressionOrganism: Komagataella phaffii CBS 7435 (fungus)

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Macromolecule #1: hLAT2

MacromoleculeName: hLAT2 / type: protein_or_peptide / ID: 1
Details: light chain of the contently linked heterodimeric amino acid transporter
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: TMAWSHPQFE KIEGRMEEGA RHRNNTEKKH PGGGESDASP EAGSGGGGVA LKKEIGLVSA CGIIVGNIIG SGIFVSPKGV LENAGSVGLA LIVWIVTGFI TVVGALCYAE LGVTIPKSGG DYSYVKDIFG GLAGFLRLWI AVLVIYPTNQ AVIALTFSNY VLQPLFPTCF ...String:
TMAWSHPQFE KIEGRMEEGA RHRNNTEKKH PGGGESDASP EAGSGGGGVA LKKEIGLVSA CGIIVGNIIG SGIFVSPKGV LENAGSVGLA LIVWIVTGFI TVVGALCYAE LGVTIPKSGG DYSYVKDIFG GLAGFLRLWI AVLVIYPTNQ AVIALTFSNY VLQPLFPTCF PPESGLRLLA AICLLLLTWV NCSSVRWATR VQDIFTAGKL LALALIIIMG IVQICKGEYF WLEPKNAFEN FQEPDIGLVA LAFLQGSFAY GGWNFLNYVT EELVDPYKNL PRAIFISIPL VTFVYVFANV AYVTAMSPQE LLASNAVAVT FGEKLLGVMA WIMPISVALS TFGGVNGSLF TSSRLFFAGA REGHLPSVLA MIHVKRCTPI PALLFTCIST LLMLVTSDMY TLINYVGFIN YLFYGVTVAG QIVLRWKKPD IPRPIKINLL FPIIYLLFWA FLLVFSLWSE PVVCGIGLAI MLTGVPVYFL GVYWQHKPKC FSDFIELLTL VSQKMCVVVY PEVERGSGTE EANEDMEEQQ QPMYQPTPTK DKDVAGQPQP

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Macromolecule #2: h4F2hc

MacromoleculeName: h4F2hc / type: protein_or_peptide / ID: 2
Details: heavy chain of the contently linked heterodimeric amino acid transporter
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Komagataella phaffii CBS 7435 (fungus)
SequenceString: TMAHHHHHHH HHGIEGRMSQ DTEVDMKEVE LNELEPEKQP MNAASGAAMS LAGAEKNGLV KIKVAEDEAE AAAAAKFTGL SKEELLKVAG SPGWVRTRWA LLLLFWLGWL GMLAGAVVII VRAPRCRELP AQKWWHTGAL YRIGDLQAFQ GHGAGNLAGL KGRLDYLSSL ...String:
TMAHHHHHHH HHGIEGRMSQ DTEVDMKEVE LNELEPEKQP MNAASGAAMS LAGAEKNGLV KIKVAEDEAE AAAAAKFTGL SKEELLKVAG SPGWVRTRWA LLLLFWLGWL GMLAGAVVII VRAPRCRELP AQKWWHTGAL YRIGDLQAFQ GHGAGNLAGL KGRLDYLSSL KVKGLVLGPI HKNQKDDVAQ TDLLQIDPNF GSKEDFDSLL QSAKKKSIRV ILDLTPNYRG ENSWFSTQVD TVATKVKDAL EFWLQAGVDG FQVRDIENLK DASSFLAEWQ NITKGFSEDR LLIAGTNSSD LQQILSLLES NKDLLLTSSY LSDSGSTGEH TKSLVTQYLN ATGNRWCSWS LSQARLLTSF LPAQLLRLYQ LMLFTLPGTP VFSYGDEIGL DAAALPGQPM EAPVMLWDES SFPDIPGAVS ANMTVKGQSE DPGSLLSLFR RLSDQRSKER SLLHGDFHAF SAGPGLFSYI RHWDQNERFL VVLNFGDVGL SAGLQASDLP ASASLPAKAD LLLSTQPGRE EGSPLELERL KLEPHEGLLL RFPYAA

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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

Concentration3 mg/mL
BufferpH: 6.8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 104971
FSC plot (resolution estimation)

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