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- PDB-8syn: Human VPS35L/VPS29/VPS26C Complex -

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Basic information

Entry
Database: PDB / ID: 8syn
TitleHuman VPS35L/VPS29/VPS26C Complex
Components
  • VPS35 endosomal protein-sorting factor-like
  • Vacuolar protein sorting-associated protein 26C
  • Vacuolar protein sorting-associated protein 29
KeywordsPROTEIN TRANSPORT / COMMD / Retriever / Commander / CCC
Function / homology
Function and homology information


WNT ligand biogenesis and trafficking / retromer, cargo-selective complex / retromer complex / Golgi to plasma membrane transport / endocytic recycling / retrograde transport, endosome to Golgi / ficolin-1-rich granule membrane / intracellular protein transport / late endosome / protein transport ...WNT ligand biogenesis and trafficking / retromer, cargo-selective complex / retromer complex / Golgi to plasma membrane transport / endocytic recycling / retrograde transport, endosome to Golgi / ficolin-1-rich granule membrane / intracellular protein transport / late endosome / protein transport / early endosome / endosome / endosome membrane / intracellular membrane-bounded organelle / Neutrophil degranulation / metal ion binding / nucleus / plasma membrane / cytosol
Similarity search - Function
VPS35 endosomal protein sorting factor-like / Vacuolar protein sorting protein 26 related / Vacuolar protein sorting-associated protein 26 / Vacuolar protein sorting-associated protein 29 / Phosphodiesterase MJ0936/Vps29 / Calcineurin-like phosphoesterase domain, lpxH-type / Calcineurin-like phosphoesterase superfamily domain / Arrestin-like, C-terminal / Metallo-dependent phosphatase-like / Immunoglobulin E-set
Similarity search - Domain/homology
Vacuolar protein sorting-associated protein 26C / VPS35 endosomal protein-sorting factor-like / Vacuolar protein sorting-associated protein 29
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsChen, Z. / Chen, B. / Burstein, E. / Han, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM128786 United States
National Science Foundation (NSF, United States)CAREER 2047640 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural organization of the retriever-CCC endosomal recycling complex.
Authors: Daniel J Boesch / Amika Singla / Yan Han / Daniel A Kramer / Qi Liu / Kohei Suzuki / Puneet Juneja / Xuefeng Zhao / Xin Long / Michael J Medlyn / Daniel D Billadeau / Zhe Chen / Baoyu Chen / Ezra Burstein /
Abstract: The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of vacuolar protein-sorting-associated protein (VPS)35L, ...The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of vacuolar protein-sorting-associated protein (VPS)35L, VPS26C and VPS29, together with the CCC complex comprising coiled-coil domain-containing (CCDC)22, CCDC93 and copper metabolism domain-containing (COMMD) proteins, plays a crucial role in this process. The precise mechanisms underlying retriever assembly and its interaction with CCC have remained elusive. Here, we present a high-resolution structure of retriever in humans determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog retromer. By combining AlphaFold predictions and biochemical, cellular and proteomic analyses, we further elucidate the structural organization of the entire retriever-CCC complex across evolution and uncover how cancer-associated mutations in humans disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with retriever-CCC-mediated endosomal recycling.
History
DepositionMay 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 13, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Dec 20, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jul 3, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: VPS35 endosomal protein-sorting factor-like
B: Vacuolar protein sorting-associated protein 29
C: Vacuolar protein sorting-associated protein 26C


Theoretical massNumber of molelcules
Total (without water)165,7883
Polymers165,7883
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein VPS35 endosomal protein-sorting factor-like / Esophageal cancer-associated protein


Mass: 109700.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VPS35L, C16orf62, 101F10.2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7Z3J2
#2: Protein Vacuolar protein sorting-associated protein 29 / hVPS29 / PEP11 homolog / Vesicle protein sorting 29


Mass: 23038.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VPS29, DC15, DC7, MDS007 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9UBQ0
#3: Protein Vacuolar protein sorting-associated protein 26C / Down syndrome critical region protein 3 / Down syndrome critical region protein A


Mass: 33049.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VPS26C, DCRA, DSCR3, DSCRA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O14972

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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

ComponentName: Trimeric Complex of full-length VPS35L/VPS29/VPS26C / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
Details: 10 mM HEPES (pH 7.0), 150 mM NaCl, 2 mM MgCl2, 2 mM DTT, and 5% (v/v) glycerol
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2150 mMsodiumn chlorideNaCl1
32 mMmagnesium chlorideMgCl21
42 mMDTTC4H10O2S21
55 %glycerolC3H8O31
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278.15 K

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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 magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3594
EM imaging opticsEnergyfilter name: GIF Quantum ER / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10PHENIX1.20.1_4487:model refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1221095
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 426624 / Symmetry type: POINT
Atomic model buildingB value: 107.9 / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410284
ELECTRON MICROSCOPYf_angle_d0.59313922
ELECTRON MICROSCOPYf_dihedral_angle_d4.3851339
ELECTRON MICROSCOPYf_chiral_restr0.0441603
ELECTRON MICROSCOPYf_plane_restr0.0051762

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