+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0161 | |||||||||
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Title | Retromer arch structures in the cell. | |||||||||
Map data | Retromer: retromer arches in cell. | |||||||||
Sample |
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Biological species | Chlamydomonas reinhardtii (plant) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 37.0 Å | |||||||||
Authors | Kovtun O / Bykov YS / Schaffer M / Engel ED / Briggs JAG | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_0161.map.gz | 953.1 KB | EMDB map data format | |
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Header (meta data) | emd-0161-v30.xml emd-0161.xml | 10.4 KB 10.4 KB | Display Display | EMDB header |
Images | emd_0161.png | 35.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0161 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0161 | HTTPS FTP |
-Related structure data
Related structure data | 0154C 0155C 0156C 0157C 0158C 0159C 0160C 0162C 0163C 5w8mC 6h7wC C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_0161.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Retromer: retromer arches in cell. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 13.69 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Whole Chlamydomonas cells
Entire | Name: Whole Chlamydomonas cells |
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Components |
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-Supramolecule #1: Whole Chlamydomonas cells
Supramolecule | Name: Whole Chlamydomonas cells / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Chlamydomonas reinhardtii (plant) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 / Details: TAP media |
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Vitrification | Cryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV Details: Blotted from the back side for 10 seconds with 10 blot force before plunging.. |
Details | C. reinhardtii cells were vitrified, and thin lamellae containing regions of the cell interior were prepared by cryo-focused ion beam milling. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 4.5 µm / Nominal magnification: 42000 |
Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.6 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Extraction | Number tomograms: 12 / Number images used: 2175 / Reference model: reference-free Method: geometrical seeding alogn the surface of manually traced tubules |
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CTF correction | Software - Name: CTFFIND (ver. 4) Details: CTF correction was performed in IMOD using defocus values measured by CTFFIND4 on non-dose-filtered images. |
Final angle assignment | Type: OTHER / Software: (Name: AV3, TOM) Details: Image processing was performed in TOM Toolbox, AV3 and Dynamo toolboxes. |
Final reconstruction | Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 37.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 553 |
Details | 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). |