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- EMDB-40884: Human VPS29/VPS35L Complex (Locally refined map) -

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Basic information

Entry
Database: EMDB / ID: EMD-40884
TitleHuman VPS29/VPS35L Complex (Locally refined map)
Map dataLocally refined map
Sample
  • Complex: Complex of full-length VPS29 with VPS35L
    • Protein or peptide: VPS35 endosomal protein-sorting factor-like
    • Protein or peptide: Vacuolar protein sorting-associated protein 29
KeywordsCOMMD / Retriever / Commander / CCC / PROTEIN TRANSPORT
Function / homology
Function and homology information


retromer, cargo-selective complex / WNT ligand biogenesis and trafficking / retromer complex / Golgi to plasma membrane transport / endocytic recycling / retrograde transport, endosome to Golgi / ficolin-1-rich granule membrane / intracellular protein transport / protein transport / late endosome ...retromer, cargo-selective complex / WNT ligand biogenesis and trafficking / retromer complex / Golgi to plasma membrane transport / endocytic recycling / retrograde transport, endosome to Golgi / ficolin-1-rich granule membrane / intracellular protein transport / protein transport / late endosome / early endosome / endosome membrane / endosome / intracellular membrane-bounded organelle / Neutrophil degranulation / metal ion binding / plasma membrane / cytosol
Similarity search - Function
VPS35 endosomal protein sorting factor-like / Vacuolar protein sorting-associated protein 29 / Phosphodiesterase MJ0936/Vps29 / Calcineurin-like phosphoesterase domain, lpxH-type / Calcineurin-like phosphoesterase superfamily domain / Metallo-dependent phosphatase-like
Similarity search - Domain/homology
VPS35 endosomal protein-sorting factor-like / Vacuolar protein sorting-associated protein 29
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChen Z / Chen B / Burstein E / Han Y
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40884.png

Downloads & links

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Map

FileDownload / File: emd_40884.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally refined map
Voxel sizeX=Y=Z: 1.0624 Å
Density
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.0017600302 - 2.0283952
Average (Standard dev.)0.00033303048 (±0.014183623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40884_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_40884_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_40884_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 : Complex of full-length VPS29 with VPS35L

EntireName: Complex of full-length VPS29 with VPS35L
Components
  • Complex: Complex of full-length VPS29 with VPS35L
    • Protein or peptide: VPS35 endosomal protein-sorting factor-like
    • Protein or peptide: Vacuolar protein sorting-associated protein 29

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Supramolecule #1: Complex of full-length VPS29 with VPS35L

SupramoleculeName: Complex of full-length VPS29 with VPS35L / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: VPS35 endosomal protein-sorting factor-like

MacromoleculeName: VPS35 endosomal protein-sorting factor-like / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 109.700453 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAVFPWHSRN RNYKAEFASC RLEAVPLEFG DYHPLKPITV TESKTKKVNR KGSTSSTSSS SSSSVVDPLS SVLDGTDPLS MFAATADPA ALAAAMDSSR RKRDRDDNSV VGSDFEPWTN KRGEILARYT TTEKLSINLF MGSEKGKAGT ATLAMSEKVR T RLEELDDF ...String:
MAVFPWHSRN RNYKAEFASC RLEAVPLEFG DYHPLKPITV TESKTKKVNR KGSTSSTSSS SSSSVVDPLS SVLDGTDPLS MFAATADPA ALAAAMDSSR RKRDRDDNSV VGSDFEPWTN KRGEILARYT TTEKLSINLF MGSEKGKAGT ATLAMSEKVR T RLEELDDF EEGSQKELLN LTQQDYVNRI EELNQSLKDA WASDQKVKAL KIVIQCSKLL SDTSVIQFYP SKFVLITDIL DT FGKLVYE RIFSMCVDSR SVLPDHFSPE NANDTAKETC LNWFFKIASI RELIPRFYVE ASILKCNKFL SKTGISECLP RLT CMIRGI GDPLVSVYAR AYLCRVGMEV APHLKETLNK NFFDFLLTFK QIHGDTVQNQ LVVQGVELPS YLPLYPPAMD WIFQ CISYH APEALLTEMM ERCKKLGNNA LLLNSVMSAF RAEFIATRSM DFIGMIKECD ESGFPKHLLF RSLGLNLALA DPPES DRLQ ILNEAWKVIT KLKNPQDYIN CAEVWVEYTC KHFTKREVNT VLADVIKHMT PDRAFEDSYP QLQLIIKKVI AHFHDF SVL FSVEKFLPFL DMFQKESVRV EVCKCIMDAF IKHQQEPTKD PVILNALLHV CKTMHDSVNA LTLEDEKRML SYLINGF IK MVSFGRDFEQ QLSFYVESRS MFCNLEPVLV QLIHSVNRLA METRKVMKGN HSRKTAAFVR ACVAYCFITI PSLAGIFT R LNLYLHSGQV ALANQCLSQA DAFFKAAISL VPEVPKMINI DGKMRPSESF LLEFLCNFFS TLLIVPDHPE HGVLFLVRE LLNVIQDYTW EDNSDEKIRI YTCVLHLLSA MSQETYLYHI DKVDSNDSLY GGDSKFLAEN NKLCETVMAQ ILEHLKTLAK DEALKRQSS LGLSFFNSIL AHGDLRNNKL NQLSVNLWHL AQRHGCADTR TMVKTLEYIK KQSKQPDMTH LTELALRLPL Q TRT

UniProtKB: VPS35 endosomal protein-sorting factor-like

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Macromolecule #2: Vacuolar protein sorting-associated protein 29

MacromoleculeName: Vacuolar protein sorting-associated protein 29 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.03832 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAGHRLVLVL GDLHIPHRCN SLPAKFKKLL VPGKIQHILC TGNLCTKESY DYLKTLAGDV HIVRGDFDEN LNYPEQKVVT VGQFKIGLI HGHQVIPWGD MASLALLQRQ FDVDILISGH THKFEAFEHE NKFYINPGSA TGAYNALETN IIPSFVLMDI Q ASTVVTYV ...String:
MAGHRLVLVL GDLHIPHRCN SLPAKFKKLL VPGKIQHILC TGNLCTKESY DYLKTLAGDV HIVRGDFDEN LNYPEQKVVT VGQFKIGLI HGHQVIPWGD MASLALLQRQ FDVDILISGH THKFEAFEHE NKFYINPGSA TGAYNALETN IIPSFVLMDI Q ASTVVTYV YQLIGDDVKV ERIEYKKPEN LYFQGGGSGG SHHHHHH

UniProtKB: Vacuolar protein sorting-associated protein 29

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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

Concentration0.25 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodiumn chloride
2.0 mMMgCl2magnesium chloride
2.0 mMC4H10O2S2DTT
5.0 %C3H8O3glycerol

Details: 10 mM HEPES (pH 7.0), 150 mM NaCl, 2 mM MgCl2, 2 mM DTT, and 5% (v/v) glycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum ER / Energy filter - Slit width: 20 eV
SoftwareName: SerialEM
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3594 / Average exposure time: 5.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 426624
Details: this is a focused refinement/local refinement from a partially refined particle stack.
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 83654
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC / Details: 3D classification (beta) from cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: Coot
RefinementSpace: REAL / Overall B value: 107.9
Output model

PDB-8sym:
Human VPS29/VPS35L Complex (Locally refined map)

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