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- EMDB-12297: Ovine rBAT ectodomain homodimer, C2-symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-12297
TitleOvine rBAT ectodomain homodimer, C2-symmetry
Map dataPostprocessed map
Sample
  • Complex: Ovine rBAT ectodomain homodimer
    • Protein or peptide: neutral and basic amino acid transport protein rBAT
  • Ligand: CALCIUM IONCalcium
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homologyOligo-1,6-glucosidase, domain 2 / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily / carbohydrate metabolic process / Glycosyl hydrolase family 13 catalytic domain-containing protein
Function and homology information
Biological speciesOvis aries (sheep)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsLee Y / Kuehlbrandt W
Funding support Germany, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
Human Frontier Science Program (HFSP)
Citation
Journal: Nat Commun / Year: 2022
Title: Ca-mediated higher-order assembly of heterodimers in amino acid transport system b biogenesis and cystinuria.
Authors: Yongchan Lee / Pattama Wiriyasermkul / Pornparn Kongpracha / Satomi Moriyama / Deryck J Mills / Werner Kühlbrandt / Shushi Nagamori /
Abstract: Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT ...Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter bAT, which constitute system b, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca mediates higher-order assembly of system b. Ca stabilizes the interface between two rBAT molecules, leading to super-dimerization of bAT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.
#1: Journal: Biorxiv / Year: 2021
Title: Ca 2+ -mediated higher-order assembly of b 0,+ AT-rBAT is a key step for system b 0,+ biogenesis and cystinuria
Authors: Lee Y / Wiriyasermkul P / Moriyama S / Mills DJ / Kuhlbrandt W / Nagamori S
History
DepositionFeb 5, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJun 1, 2022-
Current statusJun 1, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nf7
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nf7
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12297.png

Downloads & links

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Map

FileDownload / File: emd_12297.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 320 pix.
= 351.539 Å		1.1 Å/pix.
x 320 pix.
= 351.539 Å		1.1 Å/pix.
x 320 pix.
= 351.539 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09856 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.14883913 - 0.28435823
Average (Standard dev.)6.540127e-05 (±0.003373121)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 351.53918 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0985593751.0985593751.098559375
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z351.539351.539351.539
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1490.2840.000

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Supplemental data

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Mask #1

Fileemd_12297_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_12297_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_12297_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ovine rBAT ectodomain homodimer

EntireName: Ovine rBAT ectodomain homodimer
Components
  • Complex: Ovine rBAT ectodomain homodimer
    • Protein or peptide: neutral and basic amino acid transport protein rBAT
  • Ligand: CALCIUM IONCalcium
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Ovine rBAT ectodomain homodimer

SupramoleculeName: Ovine rBAT ectodomain homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Ovis aries (sheep)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 79 kDa/nm

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Macromolecule #1: neutral and basic amino acid transport protein rBAT

MacromoleculeName: neutral and basic amino acid transport protein rBAT / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Ovis aries (sheep)
Molecular weightTheoretical: 78.754281 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSAEDKSKRD SIGLNAKEGQ TNNGFVQNED ILETDLDPSS PAAGPQHNTV DILGPGEPDV KDVRPYAGMP KEVLFQFSGQ ARYRIPREV LFWLTVASVL LLIAATIAII AISPKCLDWW QAGPMYQIYP RSFRDSNKDG DGDLKGIQDK LDYITTLNIK T VWITSFYK ...String:
GSAEDKSKRD SIGLNAKEGQ TNNGFVQNED ILETDLDPSS PAAGPQHNTV DILGPGEPDV KDVRPYAGMP KEVLFQFSGQ ARYRIPREV LFWLTVASVL LLIAATIAII AISPKCLDWW QAGPMYQIYP RSFRDSNKDG DGDLKGIQDK LDYITTLNIK T VWITSFYK SSLKDFRHAV EDFQEIDPIF GTMKDFENLV AAIHDKGLKL IIDFIPNHTS DKHAWFQWSR NRTGKYTDYY IW HDCNYEN GTTIPPNNWL SVYGNSSWHF DEVRKQCYFH QFMKEQPDLN FRNPDVQEEI KEIIQFWLSK GVDGFSFNAL QYL LEAKHL RDEAQVNKTQ IPDTVTHYSQ LHHDFTTTQV GMHDIVRSFR QTMNQYSREP GRYRFMGTEA HGESITETMV YYGL PFIQE ADFPFNSYLS KLDKPSGNSV SEVITSWLEN MPEGKWPNWM TGGPDNVRLT SRLGEKYVNI MNMLVFTLPG TPITY YGEE IGMRNILAAN LNENYDTGTL FSKSPMQWDN SSNAGFSEGN HTWLPTSSDY HTVNVDVQKT QPRSALKLYQ ELSLLH ANE LLLSRGWFCY LRNDNHSIMY TRELDGINKV FLMVLNFGES SLLNLKEMIS NIPTRVRIRL STSSAYSGRE VDTHAVT LA SGEGLILEYN TGNLLHRQTA FKDRCFVSNR ACYSRVLNIL YSLC

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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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: 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

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 581697
FSC plot (resolution estimation)

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