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- EMDB-4642: 12 Angstrom structure of detergent solubilised LAT1-CD98hc -

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Basic information

Entry
Database: EMDB / ID: EMD-4642
Title12 Angstrom structure of detergent solubilised LAT1-CD98hc
Map data12 angstrom structure of LAT1-CD98hc
Sample
  • Complex: LAT1-CD98hc
    • Protein or peptide: LAT1
    • Protein or peptide: CD98hc
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsChiduza GN / Johnson R / Wright GS / Antonyuk S / Muench S / Hasnain SS
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2019
Title: LAT1 (SLC7A5) and CD98hc (SLC3A2) complex dynamics revealed by single-particle cryo-EM.
Authors: George N Chiduza / Rachel M Johnson / Gareth S A Wright / Svetlana V Antonyuk / Stephen P Muench / S Samar Hasnain /
Abstract: Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their ...Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1-CD98hc, a HAT, transports essential amino acids and drugs across the blood-brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1-CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3-3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127-130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination.
History
DepositionFeb 28, 2019-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00872
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.00872
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4642.png

Downloads & links

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Map

FileDownload / File: emd_4642.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation12 angstrom structure of LAT1-CD98hc
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.00872 / Movie #1: 0.00872
Minimum - Maximum-0.0033768932 - 0.019995214
Average (Standard dev.)0.0008652869 (±0.002896565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0030.0200.001

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Supplemental data

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Sample components

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Entire : LAT1-CD98hc

EntireName: LAT1-CD98hc
Components
  • Complex: LAT1-CD98hc
    • Protein or peptide: LAT1
    • Protein or peptide: CD98hc

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Supramolecule #1: LAT1-CD98hc

SupramoleculeName: LAT1-CD98hc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human) / Location in cell: Plasma membrane
Molecular weightTheoretical: 123 kDa/nm

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Macromolecule #1: LAT1

MacromoleculeName: LAT1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAGAGPKRRA LAAPAAEEKE EAREKMLAAK SADGSAPAGE GEGVTLQRNI TLLNGVAIIV GTIIGSGIF VTPTGVLKEA GSPGLALVVW AACGVFSIVG ALCYAELGTT ISKSGGDYAY M LEVYGSLP AFLKLWIELL IIRPSSQYIV ALVFATYLLK PLFPTCPVPE ...String:
MAGAGPKRRA LAAPAAEEKE EAREKMLAAK SADGSAPAGE GEGVTLQRNI TLLNGVAIIV GTIIGSGIF VTPTGVLKEA GSPGLALVVW AACGVFSIVG ALCYAELGTT ISKSGGDYAY M LEVYGSLP AFLKLWIELL IIRPSSQYIV ALVFATYLLK PLFPTCPVPE EAAKLVACLC VL LLTAVNC YSVKAATRVQ DAFAAAKLLA LALIILLGFV QIGKGDVSNL DPNFSFEGTK LDV GNIVLA LYSGLFAYGG WNYLNFVTEE MINPYRNLPL AIIISLPIVT LVYVLTNLAY FTTL STEQM LSSEAVAVDF GNYHLGVMSW IIPVFVGLSC FGSVNGSLFT SSRLFFVGSR EGHLP SILS MIHPQLLTPV PSLVFTCVMT LLYAFSKDIF SVINFFSFFN WLCVALAIIG MIWLRH RKP ELERPIKVNL ALPVFFILAC LFLIAVSFWK TPVECGIGFT IILSGLPVYF FGVWWKN KP KWLLQGIFST TVLCQKLMQV VPQET

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Macromolecule #2: CD98hc

MacromoleculeName: CD98hc / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MELQPPEASI AVVSIPRQLP GSHSEAGVQG LSAGDDSELG SHCVAQTGLE LLASGDPLPS ASQNAEMIE TGSDCVTQAG LQLLASSDPP ALASKNAEVT GTMSQDTEVD MKEVELNELE P EKQPMNAA SGAAMSLAGA EKNGLVKIKV AEDEAEAAAA AKFTGLSKEE ...String:
MELQPPEASI AVVSIPRQLP GSHSEAGVQG LSAGDDSELG SHCVAQTGLE LLASGDPLPS ASQNAEMIE TGSDCVTQAG LQLLASSDPP ALASKNAEVT GTMSQDTEVD MKEVELNELE P EKQPMNAA SGAAMSLAGA EKNGLVKIKV AEDEAEAAAA AKFTGLSKEE LLKVAGSPGW VR TRWALLL LFWLGWLGML AGAVVIIVRA PRCRELPAQK WWHTGALYRI GDLQAFQGHG AGN LAGLKG RLDYLSSLKV KGLVLGPIHK NQKDDVAQTD LLQIDPNFGS KEDFDSLLQS AKKK SIRVI LDLTPNYRGE NSWFSTQVDT VATKVKDALE FWLQAGVDGF QVRDIENLKD ASSFL AEWQ NITKGFSEDR LLIAGTNSSD LQQILSLLES NKDLLLTSSY LSDSGSTGEH TKSLVT QYL NATGNRWCSW SLSQARLLTS FLPAQLLRLY QLMLFTLPGT PVFSYGDEIG LDAAALP GQ PMEAPVMLWD ESSFPDIPGA VSANMTVKGQ SEDPGSLLSL FRRLSDQRSK ERSLLHGD F HAFSAGPGLF SYIRHWDQNE RFLVVLNFGD VGLSAGLQAS DLPASASLPA KADLLLSTQ PGREEGSPLE LERLKLEPHE GLLLRFPYAA

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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

Concentration2.3 mg/mL
BufferpH: 8
Details: Buffer: 100 mM Tris-Cl, 300 mM NaCl, 0.01% w/v DDM/LMNG/CHS (15:3:1), pH8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 4.96 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Details40 frames, 12 sec exposure
Startup modelType of model: INSILICO MODEL
In silico model: Generated by stochastic gradient descent in RELION 2 using a particle subset of the data.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77423
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2dh2


Chain - Chain ID: A
RefinementProtocol: RIGID BODY FIT

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