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- PDB-8dkw: Cryo-EM structure of cystinosin N288K mutant in a cytosol-open st... -

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Basic information

Entry
Database: PDB / ID: 8dkw
TitleCryo-EM structure of cystinosin N288K mutant in a cytosol-open state at pH5.0
Components
  • Fab 3H5 Heavy Chain
  • Fab 3H5 Kappa Chain
  • Isoform 2 of Cystinosin
KeywordsMEMBRANE PROTEIN/Transport protein / Cystine / 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 speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsSchmiege, P. / Li, X.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 12, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Fab 3H5 Heavy Chain
B: Fab 3H5 Kappa Chain
P: Isoform 2 of Cystinosin


Theoretical massNumber of molelcules
Total (without water)98,3323
Polymers98,3323
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody Fab 3H5 Heavy Chain


Mass: 26768.107 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#2: Antibody Fab 3H5 Kappa Chain


Mass: 25468.404 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#3: Protein Isoform 2 of Cystinosin


Mass: 46095.434 Da / Num. of mol.: 1 / Mutation: N288K, T260I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CTNS / Production host: Homo sapiens (human) / References: UniProt: O60931
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cystinosin N288K - Fab 3H5 complex at pH5.0COMPLEXall0RECOMBINANT
2Cystinosin N288KCOMPLEX#31RECOMBINANT
3Fab 3H5COMPLEX#1-#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Buffer solutionpH: 5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 61.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 509919 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0064489
ELECTRON MICROSCOPYf_angle_d0.6346121
ELECTRON MICROSCOPYf_dihedral_angle_d2.9532576
ELECTRON MICROSCOPYf_chiral_restr0.047693
ELECTRON MICROSCOPYf_plane_restr0.004764

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