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- PDB-6h7w: Model of retromer-Vps5 complex assembled on membrane. -

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Basic information

Entry
Database: PDB / ID: 6h7w
TitleModel of retromer-Vps5 complex assembled on membrane.
Components
  • (Putative vacuolar protein sorting-associated ...Vacuole) x 3
  • (Vacuolar protein sorting-associated protein ...Vacuole) x 3
KeywordsPROTEIN TRANSPORT / retromer / endosome / sorting nexin / BAR / membrane trafficking
Function / homology
Function and homology information


ascospore-type prospore membrane formation / prospore membrane / retromer, cargo-selective complex / retromer complex / retrograde transport, endosome to Golgi / phosphatidylinositol binding / intracellular protein transport / protein transport / endosome / identical protein binding / cytosol
Similarity search - Function
Vacuolar protein sorting-associated protein 5-like / Sorting nexin Vps5, BAR domain / Sorting nexin Vps5-like, C-terminal / Vps5 C terminal like / Vacuolar protein sorting protein 26 related / Vacuolar protein sorting-associated protein 26 / Vacuolar protein sorting-associated protein 35 / Vacuolar protein sorting-associated protein 35, C-terminal / Vacuolar protein sorting-associated protein 35 / Vacuolar protein sorting-associated protein 29 ...Vacuolar protein sorting-associated protein 5-like / Sorting nexin Vps5, BAR domain / Sorting nexin Vps5-like, C-terminal / Vps5 C terminal like / Vacuolar protein sorting protein 26 related / Vacuolar protein sorting-associated protein 26 / Vacuolar protein sorting-associated protein 35 / Vacuolar protein sorting-associated protein 35, C-terminal / Vacuolar protein sorting-associated protein 35 / Vacuolar protein sorting-associated protein 29 / Phosphodiesterase MJ0936/Vps29 / Calcineurin-like phosphoesterase domain, lpxH-type / Calcineurin-like phosphoesterase superfamily domain / Arrestin-like, C-terminal / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / AH/BAR domain superfamily / Metallo-dependent phosphatase-like
Similarity search - Domain/homology
Vacuolar protein sorting-associated protein 29 / Vacuolar protein sorting-associated protein 26-like protein / Vacuolar protein sorting-associated protein 35 / Putative vacuolar protein sorting-associated protein
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 11.4 Å
AuthorsKovtun, O. / Leneva, N. / Ariotti, N. / Rohan, T.S. / Owen, D.J. / Briggs, J.A.G. / Collins, B.M.
Funding support United Kingdom, Australia, 7items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/16 United Kingdom
Wellcome Trust207455/Z/17/Z United Kingdom
Australian Research CouncilDP160101743 Australia
National Health and Medical Research Council (Australia)APP1042082 Australia
National Health and Medical Research Council (Australia)APP1058734 Australia
National Health and Medical Research Council (Australia)APP1041929 Australia
National Health and Medical Research Council (Australia)APP1136021 Australia
CitationJournal: Nature / Year: 2018
Title: Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.
Authors: Oleksiy Kovtun / Natalya Leneva / Yury S Bykov / Nicholas Ariotti / Rohan D Teasdale / Miroslava Schaffer / Benjamin D Engel / David J Owen / John A G Briggs / Brett M Collins /
Abstract: Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular ...Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.
History
DepositionJul 31, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 26, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 10, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
J: Vacuolar protein sorting-associated protein 26-like protein
C: Vacuolar protein sorting-associated protein 26-like protein
B: Putative vacuolar protein sorting-associated protein
A: Putative vacuolar protein sorting-associated protein
D: Putative vacuolar protein sorting-associated protein
G: Putative vacuolar protein sorting-associated protein
E: Putative vacuolar protein sorting-associated protein
K: Putative vacuolar protein sorting-associated protein
O: Putative vacuolar protein sorting-associated protein
P: Putative vacuolar protein sorting-associated protein
M: Putative vacuolar protein sorting-associated protein
N: Putative vacuolar protein sorting-associated protein
H: Putative vacuolar protein sorting-associated protein
F: Putative vacuolar protein sorting-associated protein
V: Putative vacuolar protein sorting-associated protein
L: Putative vacuolar protein sorting-associated protein
S: Vacuolar protein sorting-associated protein 29
Q: Vacuolar protein sorting-associated protein 35
T: Vacuolar protein sorting-associated protein 29
R: Vacuolar protein sorting-associated protein 35


Theoretical massNumber of molelcules
Total (without water)750,87220
Polymers750,87220
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Retromer core and Vps5 assemble in solution and can be isolated in heterogenous peak migrating as >1MDa complex on Superose 6 SEC.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Vacuolar protein sorting-associated protein ... , 3 types, 6 molecules JCSTQR

#1: Protein Vacuolar protein sorting-associated protein 26-like protein / Vacuole


Mass: 34308.449 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0009750 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S0E6
#5: Protein Vacuolar protein sorting-associated protein 29 / Vacuole


Mass: 21470.654 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0002370 / Production host: Escherichia coli (E. coli) / References: UniProt: G0RZB5
#6: Protein Vacuolar protein sorting-associated protein 35 / Vacuole


Mass: 96140.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0035730 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S709

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Putative vacuolar protein sorting-associated ... , 3 types, 14 molecules BAGEPNHFDKVLOM

#2: Protein
Putative vacuolar protein sorting-associated protein / Vacuole


Mass: 42060.762 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0068290 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SH11
#3: Protein
Putative vacuolar protein sorting-associated protein / Vacuole


Mass: 14897.982 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0068290 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SH11
#4: Protein Putative vacuolar protein sorting-associated protein / Vacuole


Mass: 25477.904 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0068290 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SH11

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Retromer-Vps5 complex assembled on the membrane. / Type: COMPLEX
Details: Vps26/Vps35/Vps29 trimer recruited to the membrane via Vps5 PX-BAR protein.
Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-KOHC8H19KN2O5S1
2200 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The solution-assembled complex was incubated with Folch liposomes at room temperature.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 292 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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 6500 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 3.17 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2
Details: Tomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 10

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Processing

EM software
IDNameVersionCategory
2SerialEM4.8image acquisition
4CTFFIND4CTF correction
5IMODCTF correction
8UCSF Chimeramodel fitting
11AV3final Euler assignment
12TOMfinal Euler assignment
15NAMDmodel refinement
Image processingDetails: After motion correction in "alignframes" (IMOD), each of the images in the tilt series was low-pass filtered according to the electron-dose acquired by the sample (Grant and Grigorieff, 2015).
CTF correctionDetails: CTF correction was performed with ctfphaseflip IMOD command using defocus values measured by CTFFIND4 on non-dose-filtered images.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 11.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16037 / Algorithm: BACK PROJECTION / Symmetry type: POINT
EM volume selectionMethod: geometrical seeding alogn the surface of manually traced tubules
Num. of tomograms: 71 / Num. of volumes extracted: 194885 / Reference model: reference-free
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Note that the associated PDB model was not generated by fitting into this map! It was generated by fitting into the associated local high-resolution maps and then combined. Initial global ...Details: Note that the associated PDB model was not generated by fitting into this map! It was generated by fitting into the associated local high-resolution maps and then combined. Initial global ridgid body fitting was done using Chimera, followed by MDFF within NAMD for flexible fitting. Note that side chain positions are not reliable in model generated by refinement into a map at this resolution.

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