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- EMDB-15210: Human 4F2hc-LAT2 heterodimeric amino acid transporter in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-15210
TitleHuman 4F2hc-LAT2 heterodimeric amino acid transporter in complex with anticalin D11vs
Map datahuman 4F2hc-LAT2 in complex to anticalin D11vs
Sample
  • Complex: Heterodimeric amino acid transporter 4F2hc-LAT2 in complex with anticalin D11vs
    • Protein or peptide: 4F2hc cell-surface antigen heavy chain
    • Protein or peptide: Large neutral amino acids transporter small subunit 2
    • Protein or peptide: Anticalin D11vs
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
Function / homology
Function and homology information


proline transmembrane transport / glycine transmembrane transporter activity / organic cation transmembrane transporter activity / thyroid hormone transmembrane transporter activity / glycine transport / amino acid import across plasma membrane / L-amino acid transmembrane transporter activity / : / L-leucine import across plasma membrane / L-alanine transmembrane transporter activity ...proline transmembrane transport / glycine transmembrane transporter activity / organic cation transmembrane transporter activity / thyroid hormone transmembrane transporter activity / glycine transport / amino acid import across plasma membrane / L-amino acid transmembrane transporter activity / : / L-leucine import across plasma membrane / L-alanine transmembrane transporter activity / L-alanine import across plasma membrane / valine transport / L-leucine transmembrane transporter activity / amino acid transmembrane transport / L-leucine transport / thyroid hormone transport / neutral amino acid transport / toxin transmembrane transporter activity / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / antiporter activity / amino acid transport / Basigin interactions / microvillus membrane / tryptophan transport / transport across blood-brain barrier / basal plasma membrane / peptide antigen binding / basolateral plasma membrane / apical plasma membrane / protein heterodimerization activity / plasma membrane
Similarity search - Function
L-type amino acid transporter / Amino acid/polyamine transporter I / Amino acid permease
Similarity search - Domain/homology
Large neutral amino acids transporter small subunit 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsJeckelmann JM / Fotiadis D
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science FoundationNCCR Transcure Switzerland
CitationJournal: Sci Rep / Year: 2022
Title: Structure of the human heterodimeric transporter 4F2hc-LAT2 in complex with Anticalin, an alternative binding protein for applications in single-particle cryo-EM.
Authors: Jean-Marc Jeckelmann / Thomas Lemmin / Martin Schlapschy / Arne Skerra / Dimitrios Fotiadis /
Abstract: Cryo-EM structure determination of relatively small and flexible membrane proteins at high resolution is challenging. Increasing the size and structural features by binding of high affinity proteins ...Cryo-EM structure determination of relatively small and flexible membrane proteins at high resolution is challenging. Increasing the size and structural features by binding of high affinity proteins to the biomolecular target allows for better particle alignment and may result in structural models of higher resolution and quality. Anticalins are alternative binding proteins to antibodies, which are based on the lipocalin scaffold and show potential for theranostic applications. The human heterodimeric amino acid transporter 4F2hc-LAT2 is a membrane protein complex that mediates transport of certain amino acids and derivatives thereof across the plasma membrane. Here, we present and discuss the cryo-EM structure of human 4F2hc-LAT2 in complex with the anticalin D11vs at 3.2 Å resolution. Relative high local map resolution (2.8-3.0 Å) in the LAT2 substrate binding site together with molecular dynamics simulations indicated the presence of fixed water molecules potentially involved in shaping and stabilizing this region. Finally, the presented work expands the application portfolio of anticalins and widens the toolset of binding proteins to promote high-resolution structure solution by single-particle cryo-EM.
History
DepositionJun 18, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateNov 9, 2022-
Current statusNov 9, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15210.png

Downloads & links

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Map

FileDownload / File: emd_15210.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman 4F2hc-LAT2 in complex to anticalin D11vs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 256 pix.
= 210.432 Å		0.82 Å/pix.
x 256 pix.
= 210.432 Å		0.82 Å/pix.
x 256 pix.
= 210.432 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.1042461 - 1.7155521
Average (Standard dev.)0.0032358086 (±0.038179364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Halve map B

Fileemd_15210_half_map_1.map
AnnotationHalve map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halve map A

Fileemd_15210_half_map_2.map
AnnotationHalve map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heterodimeric amino acid transporter 4F2hc-LAT2 in complex with a...

EntireName: Heterodimeric amino acid transporter 4F2hc-LAT2 in complex with anticalin D11vs
Components
  • Complex: Heterodimeric amino acid transporter 4F2hc-LAT2 in complex with anticalin D11vs
    • Protein or peptide: 4F2hc cell-surface antigen heavy chain
    • Protein or peptide: Large neutral amino acids transporter small subunit 2
    • Protein or peptide: Anticalin D11vs
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: Heterodimeric amino acid transporter 4F2hc-LAT2 in complex with a...

SupramoleculeName: Heterodimeric amino acid transporter 4F2hc-LAT2 in complex with anticalin D11vs
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant strain: KM71H
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: 4F2hc cell-surface antigen heavy chain

MacromoleculeName: 4F2hc 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: 60.063934 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: TMAHHHHHHH HHGIEGRMSQ DTEVDMKEVE LNELEPEKQP MNAASGAAMS LAGAEKNGLV KIKVAEDEAE AAAAAKFTGL SKEELLKVA GSPGWVRTRW ALLLLFWLGW LGMLAGAVVI IVRAPRCREL PAQKWWHTGA LYRIGDLQAF QGHGAGNLAG L KGRLDYLS ...String:
TMAHHHHHHH HHGIEGRMSQ DTEVDMKEVE LNELEPEKQP MNAASGAAMS LAGAEKNGLV KIKVAEDEAE AAAAAKFTGL SKEELLKVA GSPGWVRTRW ALLLLFWLGW LGMLAGAVVI IVRAPRCREL PAQKWWHTGA LYRIGDLQAF QGHGAGNLAG L KGRLDYLS SLKVKGLVLG PIHKNQKDDV AQTDLLQIDP NFGSKEDFDS LLQSAKKKSI RVILDLTPNY RGENSWFSTQ VD TVATKVK DALEFWLQAG VDGFQVRDIE NLKDASSFLA EWQNITKGFS EDRLLIAGTN SSDLQQILSL LESNKDLLLT SSY LSDSGS TGEHTKSLVT QYLNATGNRW CSWSLSQARL LTSFLPAQLL RLYQLMLFTL PGTPVFSYGD EIGLDAAALP GQPM EAPVM LWDESSFPDI PGAVSANMTV KGQSEDPGSL LSLFRRLSDQ RSKERSLLHG DFHAFSAGPG LFSYIRHWDQ NERFL VVLN FGDVGLSAGL QASDLPASAS LPAKADLLLS TQPGREEGSP LELERLKLEP HEGLLLRFPY AA

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Macromolecule #2: Large neutral amino acids transporter small subunit 2

MacromoleculeName: Large neutral amino acids transporter small subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.121078 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MAWSHPQFEK IEGRMEEGAR HRNNTEKKHP GGGESDASPE AGSGGGGVAL KKEIGLVSAC GIIVGNIIGS GIFVSPKGVL ENAGSVGLA LIVWIVTGFI TVVGALCYAE LGVTIPKSGG DYSYVKDIFG GLAGFLRLWI AVLVIYPTNQ AVIALTFSNY V LQPLFPTC ...String:
MAWSHPQFEK IEGRMEEGAR HRNNTEKKHP GGGESDASPE AGSGGGGVAL KKEIGLVSAC GIIVGNIIGS GIFVSPKGVL ENAGSVGLA LIVWIVTGFI TVVGALCYAE LGVTIPKSGG DYSYVKDIFG GLAGFLRLWI AVLVIYPTNQ AVIALTFSNY V LQPLFPTC FPPESGLRLL AAICLLLLTW VNCSSVRWAT RVQDIFTAGK LLALALIIIM GIVQICKGEY FWLEPKNAFE NF QEPDIGL VALAFLQGSF AYGGWNFLNY VTEELVDPYK NLPRAIFISI PLVTFVYVFA NVAYVTAMSP QELLASNAVA VTF GEKLLG VMAWIMPISV ALSTFGGVNG SLFTSSRLFF AGAREGHLPS VLAMIHVKRC TPIPALLFTC ISTLLMLVTS DMYT LINYV GFINYLFYGV TVAGQIVLRW KKPDIPRPIK INLLFPIIYL LFWAFLLVFS LWSEPVVCGI GLAIMLTGVP VYFLG VYWQ HKPKCFSDFI ELLTLVSQKM CVVVYPEVER GSGTEEANED MEEQQQPMYQ PTPTKDKDVA GQPQP

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Macromolecule #3: Anticalin D11vs

MacromoleculeName: Anticalin D11vs / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.327084 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QDSTSDLIPA PPLSKVPLQQ NFQDNQFHGK WYVVGRAGNT GLREDKDPGK MFATIYELKE DKSYNVTYVW SGQKKCMYSI VTFVPGSQP GEFTLGNIKS APGRTSWLVR VVSTNYNQHA MVFFKSVTQN REGFAITLYG RTKELTSELK ENFIRFSKSL G LPENHIVF PVPIDQCIDG SAWSHPQFEK

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 3 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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: 8
Component:
ConcentrationName
20.0 mMBis-Tris-propane
150.0 mMNaClSodium chloride
0.02 %GDN
1.0 %glycerol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.023 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: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14)
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 552926
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8a6l:
Human 4F2hc-LAT2 heterodimeric amino acid transporter in complex with anticalin D11vs

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