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- EMDB-27493: Cryo-EM structure of cystinosin N288K mutant in a cytosol-open st... -

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Basic information

Entry
Database: EMDB / ID: EMD-27493
TitleCryo-EM structure of cystinosin N288K mutant in a cytosol-open state at pH7.5
Map dataCystinosin N288K mutant at pH7.5
Sample
  • Complex: Cystinosin N288K - Fab 3H5 complex at pH7.5
    • Complex: Cystinosin N288K
      • Protein or peptide: Isoform 2 of Cystinosin
    • Complex: Fab 3H5
      • Protein or peptide: Fab 3H5 Heavy Chain
      • Protein or peptide: Fab 3H5 Kappa Chain
KeywordsCystine / transporter / lysosome / MEMBRANE PROTEIN / MEMBRANE PROTEIN-Transport protein complex
Function / homology
Function and homology information


proximal tubule morphogenesis / regulation of melanin biosynthetic process / solute:proton symporter activity / positive regulation of thyroid hormone generation / L-cystine transmembrane transporter activity / renal water absorption / brush border assembly / L-cystine transport / renal phosphate ion absorption / renal D-glucose absorption ...proximal tubule morphogenesis / regulation of melanin biosynthetic process / solute:proton symporter activity / positive regulation of thyroid hormone generation / L-cystine transmembrane transporter activity / renal water absorption / brush border assembly / L-cystine transport / renal phosphate ion absorption / renal D-glucose absorption / Transport of inorganic cations/anions and amino acids/oligopeptides / negative regulation of hydrogen peroxide biosynthetic process / Miscellaneous transport and binding events / regulation of TORC1 signaling / melanin biosynthetic process / grooming behavior / renal albumin absorption / melanosome membrane / amino acid metabolic process / adult walking behavior / lens development in camera-type eye / positive regulation of mitochondrial membrane potential / thyroid gland development / long-term memory / monoatomic ion transport / ATP metabolic process / glutathione metabolic process / positive regulation of TORC1 signaling / brain development / visual learning / transmembrane transport / cognition / late endosome / melanosome / protein transport / lysosome / lysosomal membrane / intracellular membrane-bounded organelle / negative regulation of apoptotic process / extracellular exosome / plasma membrane
Similarity search - Function
Lysosomal cystine transporter / Repeated motif present between transmembrane helices in cystinosin, yeast ERS1p, mannose-P-dolichol utilization defect 1, and other hypothetical proteins. / PQ-loop repeat / PQ loop repeat
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSchmiege P / Li X
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
Welch Foundation United States
CitationJournal: Cell / Year: 2022
Title: Structure and mechanism of human cystine exporter cystinosin.
Authors: Xue Guo / Philip Schmiege / Tufa E Assafa / Rong Wang / Yan Xu / Linda Donnelly / Michael Fine / Xiaodan Ni / Jiansen Jiang / Glenn Millhauser / Liang Feng / Xiaochun Li /
Abstract: Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of ...Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporter's conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy.
History
DepositionJul 6, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27493.png

Downloads & links

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Map

FileDownload / File: emd_27493.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCystinosin N288K mutant at pH7.5
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.748
Minimum - Maximum-2.9359095 - 4.5469775
Average (Standard dev.)0.0036680573 (±0.083564736)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_27493_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_27493_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 : Cystinosin N288K - Fab 3H5 complex at pH7.5

EntireName: Cystinosin N288K - Fab 3H5 complex at pH7.5
Components
  • Complex: Cystinosin N288K - Fab 3H5 complex at pH7.5
    • Complex: Cystinosin N288K
      • Protein or peptide: Isoform 2 of Cystinosin
    • Complex: Fab 3H5
      • Protein or peptide: Fab 3H5 Heavy Chain
      • Protein or peptide: Fab 3H5 Kappa Chain

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Supramolecule #1: Cystinosin N288K - Fab 3H5 complex at pH7.5

SupramoleculeName: Cystinosin N288K - Fab 3H5 complex at pH7.5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Cystinosin N288K

SupramoleculeName: Cystinosin N288K / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Fab 3H5

SupramoleculeName: Fab 3H5 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Fab 3H5 Heavy Chain

MacromoleculeName: Fab 3H5 Heavy Chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.768107 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSE VMLVESGGGL VKPGGSLKLS CAASGFTFSN YAMSWVRQTP EKRLEWVAAI SGNEGTYTYY PDSVRGRFT ISRDNARNNL YLQISSLRSE DTALYYCARY GLVGALDFWG QGASVTVSSA STKGPSVFPL APSSKSTSGG T AALGCLVK ...String:
MGWSCIILFL VATATGVHSE VMLVESGGGL VKPGGSLKLS CAASGFTFSN YAMSWVRQTP EKRLEWVAAI SGNEGTYTYY PDSVRGRFT ISRDNARNNL YLQISSLRSE DTALYYCARY GLVGALDFWG QGASVTVSSA STKGPSVFPL APSSKSTSGG T AALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KS CDKTHHH HHH

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Macromolecule #2: Fab 3H5 Kappa Chain

MacromoleculeName: Fab 3H5 Kappa Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.468404 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSD IQMNQSPSTL SASLGDTITI TCRASQNIDV WLNWYQQKPG DIPKLLIYEA SNLHTGVPSR FSGSGSGTD FTLAISSLQP EDIATYYCLQ GQDYPFTFGS GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY P REAKVQWK ...String:
MGWSCIILFL VATATGVHSD IQMNQSPSTL SASLGDTITI TCRASQNIDV WLNWYQQKPG DIPKLLIYEA SNLHTGVPSR FSGSGSGTD FTLAISSLQP EDIATYYCLQ GQDYPFTFGS GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY P REAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

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Macromolecule #3: Isoform 2 of Cystinosin

MacromoleculeName: Isoform 2 of Cystinosin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.095434 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MIRNWLTIFI LFPLKLVEKC ESSVSLTVPP VVKLENGSST NVSLTLRPPL NATLVITFEI TFRSKNITIL ELPDEVVVPP GVTNSSFQV TSQNVGQLTV YLHGNHSNQT GPRIRFLVIR SSAISIINQV IGWIYFVAWS ISFYPQVIMN WRRKSVIGLS F DFVALNLT ...String:
MIRNWLTIFI LFPLKLVEKC ESSVSLTVPP VVKLENGSST NVSLTLRPPL NATLVITFEI TFRSKNITIL ELPDEVVVPP GVTNSSFQV TSQNVGQLTV YLHGNHSNQT GPRIRFLVIR SSAISIINQV IGWIYFVAWS ISFYPQVIMN WRRKSVIGLS F DFVALNLT GFVAYSVFNI GLLWVPYIKE QFLLKYPNGV NPVNSNDVFF SLHAVVLTLI IIVQCCLYER GGQRVSWPAI GF LVLAWLF AFVTMIVAAV GVITWLQFLF CFSYIKLAVT LVKYFPQAYM KFYYKSTEGW SIGNVLLDFT GGSFSLLQMF LQS YNNDQW TLIFGDPTKF GLGVFSIVFD VVFFIQHFCL YRKRPGLQAA RTGSGSRLRQ DWAPSLQPKA LPQTTSVSAS SLKG DYKDD DDK

UniProtKB: Cystinosin

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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: 5
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: 61.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 323738
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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