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Yorodumi- EMDB-0154: Retromer-Vps5: low-resolution overview map centred on the Vps26 dimer. -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0154 | ||||||||||||||||||||||||
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Title | Retromer-Vps5: low-resolution overview map centred on the Vps26 dimer. | ||||||||||||||||||||||||
Map data | Low-resolution overview map of retromer-Vps5 centered on the Vps26 dimer. | ||||||||||||||||||||||||
Sample |
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Keywords | retromer / endosome / sorting nexin / BAR / membrane trafficking / PROTEIN TRANSPORT | ||||||||||||||||||||||||
Function / homology | Function and homology information retromer, cargo-selective complex / retromer complex / retrograde transport, endosome to Golgi / phosphatidylinositol binding / intracellular protein transport / protein transport / identical protein binding / cytosol Similarity search - Function | ||||||||||||||||||||||||
Biological species | Chaetomium thermophilum var. thermophilum DSM 1495 (fungus) / Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) | ||||||||||||||||||||||||
Method | subtomogram averaging / cryo EM / Resolution: 11.4 Å | ||||||||||||||||||||||||
Authors | Kovtun O / Leneva N / Ariotti N / Rohan TS / Owen DJ / Briggs JAG / Collins BM | ||||||||||||||||||||||||
Funding support | United Kingdom, Australia, 7 items
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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_0154.map.gz | 10.6 MB | EMDB map data format | |
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Header (meta data) | emd-0154-v30.xml emd-0154.xml | 22.6 KB 22.6 KB | Display Display | EMDB header |
Images | emd_0154.png | 114.1 KB | ||
Filedesc metadata | emd-0154.cif.gz | 7.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0154 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0154 | HTTPS FTP |
-Validation report
Summary document | emd_0154_validation.pdf.gz | 246.9 KB | Display | EMDB validaton report |
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Full document | emd_0154_full_validation.pdf.gz | 246 KB | Display | |
Data in XML | emd_0154_validation.xml.gz | 5.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0154 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0154 | HTTPS FTP |
-Related structure data
Related structure data | 6h7wMC 0155C 0156C 0157C 0158C 0159C 0160C 0161C 0162C 0163C 5w8mC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_0154.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Low-resolution overview map of retromer-Vps5 centered on the Vps26 dimer. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.7 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 : Retromer-Vps5 complex assembled on the membrane.
Entire | Name: Retromer-Vps5 complex assembled on the membrane. |
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Components |
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-Supramolecule #1: Retromer-Vps5 complex assembled on the membrane.
Supramolecule | Name: Retromer-Vps5 complex assembled on the membrane. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Vps26/Vps35/Vps29 trimer recruited to the membrane via Vps5 PX-BAR protein. |
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Source (natural) | Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus) |
-Macromolecule #1: Vacuolar protein sorting-associated protein 26-like protein
Macromolecule | Name: Vacuolar protein sorting-associated protein 26-like protein type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) Strain: DSM 1495 / CBS 144.50 / IMI 039719 |
Molecular weight | Theoretical: 34.308449 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: FSTPVDIDIV LADADKRAMV DVKLDKNRRE KVPLYMDGES VKGCVTVRPK DGKRLEHTGI KVQFIGTIEM FFDRGNHYEF LSLVQELAA PGELQHPQTF DFNFKNVEKQ YESYNGINVK LRYFVRVTVS RRMADVIREK DIWVYSYRIP PELNSSIKMD V GIEDCLHI ...String: FSTPVDIDIV LADADKRAMV DVKLDKNRRE KVPLYMDGES VKGCVTVRPK DGKRLEHTGI KVQFIGTIEM FFDRGNHYEF LSLVQELAA PGELQHPQTF DFNFKNVEKQ YESYNGINVK LRYFVRVTVS RRMADVIREK DIWVYSYRIP PELNSSIKMD V GIEDCLHI EFEYSKSKYH LKDVIVGRIY FLLVRLKIKH MELSIIRRET TGVAPNQYNE SETLVRFEIM DGSPSRGETI PI RLFLGGF DLTPTFRDVN KKFSTRYYLS LVLIDEDARR YFKQSEIILY RQPPE UniProtKB: Vacuolar protein sorting-associated protein 26-like protein |
-Macromolecule #2: Putative vacuolar protein sorting-associated protein
Macromolecule | Name: Putative vacuolar protein sorting-associated protein / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO |
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Source (natural) | Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) Strain: DSM 1495 / CBS 144.50 / IMI 039719 |
Molecular weight | Theoretical: 42.060762 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: ARPTFHITVG DPHKVGDLAT SHIVYSVRTK TTSKAYKQPE FEVKRRYRDF LWLYNTLHSN NPGVVVPPPP EKQAVGRFES NFVESRRAA LEKMLNKIAA HPTLQLDADL KLFLESESFN IDVKHKERKE PPLGESKGVF GSLGFGGGGN KFVEQDDWFH D RRVYLDAL ...String: ARPTFHITVG DPHKVGDLAT SHIVYSVRTK TTSKAYKQPE FEVKRRYRDF LWLYNTLHSN NPGVVVPPPP EKQAVGRFES NFVESRRAA LEKMLNKIAA HPTLQLDADL KLFLESESFN IDVKHKERKE PPLGESKGVF GSLGFGGGGN KFVEQDDWFH D RRVYLDAL ENQLKALLKA MDNMVAQRKA MAEAAADFSA SLHALSTVEL SPTLSGPLDA LSELQLAIRD VYERQAQQDV LT FGIIIEE YIRLIGSVKQ AFSQRQKAFH SWHSAESELM KKKAAQDKLL RQGKTQQDRL NQVNAEVIDA ERKVHQARLL FED MGRLLR SELDRFEREK VEDFKSGVET FLESAVEAQK ELIEKWETFL MQ UniProtKB: Putative vacuolar protein sorting-associated protein |
-Macromolecule #3: Putative vacuolar protein sorting-associated protein
Macromolecule | Name: Putative vacuolar protein sorting-associated protein / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) Strain: DSM 1495 / CBS 144.50 / IMI 039719 |
Molecular weight | Theoretical: 14.897982 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: ARPTFHITVG DPHKVGDLAT SHIVYSVRTK TTSKAYKQPE FEVKRRYRDF LWLYNTLHSN NPGVVVPPPP EKQAVGRFES NFVESRRAA LEKMLNKIAA HPTLQLDADL KLFLESESFN IDVKHKERKE UniProtKB: Putative vacuolar protein sorting-associated protein |
-Macromolecule #4: Putative vacuolar protein sorting-associated protein
Macromolecule | Name: Putative vacuolar protein sorting-associated protein / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) Strain: DSM 1495 / CBS 144.50 / IMI 039719 |
Molecular weight | Theoretical: 25.477904 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: NKFVEQDDWF HDRRVYLDAL ENQLKALLKA MDNMVAQRKA MAEAAADFSA SLHALSTVEL SPTLSGPLDA LSELQLAIRD VYERQAQQD VLTFGIIIEE YIRLIGSVKQ AFSQRQKAFH SWHSAESELM KKKAAQDKLL RQGKTQQDRL NQVNAEVIDA E RKVHQARL ...String: NKFVEQDDWF HDRRVYLDAL ENQLKALLKA MDNMVAQRKA MAEAAADFSA SLHALSTVEL SPTLSGPLDA LSELQLAIRD VYERQAQQD VLTFGIIIEE YIRLIGSVKQ AFSQRQKAFH SWHSAESELM KKKAAQDKLL RQGKTQQDRL NQVNAEVIDA E RKVHQARL LFEDMGRLLR SELDRFEREK VEDFKSGVET FLESAVEAQK ELIEKWETFL MQ UniProtKB: Putative vacuolar protein sorting-associated protein |
-Macromolecule #5: Vacuolar protein sorting-associated protein 29
Macromolecule | Name: Vacuolar protein sorting-associated protein 29 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) Strain: DSM 1495 / CBS 144.50 / IMI 039719 |
Molecular weight | Theoretical: 21.470654 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: AFLILVIGNL HIPDRALDIP PKFKKLLSPG KISQTLCLGN LTDRATYDYL RSISPDLKIV RGRMDVEATS LPLMQVVTHG SLRIGFLEG FTLVSEEPDV LLAEANKLDV DVLCWAGGSH RFECFEYMDK FFVNPGSATG AFTTDWLAEG EEVVPSFCLM D VQGISLTL ...String: AFLILVIGNL HIPDRALDIP PKFKKLLSPG KISQTLCLGN LTDRATYDYL RSISPDLKIV RGRMDVEATS LPLMQVVTHG SLRIGFLEG FTLVSEEPDV LLAEANKLDV DVLCWAGGSH RFECFEYMDK FFVNPGSATG AFTTDWLAEG EEVVPSFCLM D VQGISLTL YVYQLRKDEN GTENVAVEKV TYTKP UniProtKB: Vacuolar protein sorting-associated protein 29 |
-Macromolecule #6: Vacuolar protein sorting-associated protein 35
Macromolecule | Name: Vacuolar protein sorting-associated protein 35 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus) Strain: DSM 1495 / CBS 144.50 / IMI 039719 |
Molecular weight | Theoretical: 96.140016 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: RLLEDALIAV RQQTAMMRKF LDTPGKLMDA LKCCSTLVSE LRTSSLSPKQ YYELYMAVFD ALRYLSAHLR ENHPVNHLAD LYELVQYAG NIIPRLYLMI TVGTAYMSID GAPVKELMKD MMDMSRGVQH PVRGLFLRYY LSGQARDYLP TGDSDGPEGN L QDSINFIL ...String: RLLEDALIAV RQQTAMMRKF LDTPGKLMDA LKCCSTLVSE LRTSSLSPKQ YYELYMAVFD ALRYLSAHLR ENHPVNHLAD LYELVQYAG NIIPRLYLMI TVGTAYMSID GAPVKELMKD MMDMSRGVQH PVRGLFLRYY LSGQARDYLP TGDSDGPEGN L QDSINFIL TNFVEMNKLW VRLQHQGHSR ERDLRTQERR ELQLLVGSNI VRLSQLVDLP TYRDSILGPL LEQIVQCRDI LA QEYLLEV ITQVFPDEYH LHTLDQFLGA VSRLNPHVNV KAIVIGMMNR LSDYAERESQ NEPEEDRAKL EEEALAKLLE KTK LGQNSE LEPQNGDHPD TEVSSTTDSA QAPSTADSDT TAVNGEEEPV RKRRGIPVNV PLYDIFFDQV QHLVQAQHLP IQDT IALCC SLANLSLNIY PERLDYVDGI LAYALAKVKE HANSADLHSQ PAQQSLLSLL QSPLRRYVSI FTALSLPTYV SLFQA QTYP TRRAIAGEIV RTLLKNQTLI STPAHLENVL EILKVLIKEG SQPPAGYPGV VQPRARPLET DETMEEQGWL ARLVHL IHS DDNDTQFRLL QMTRKAYAEG NERIRTTTPP LITAGLKLAR RFKAREHYDD NWSSQSSSLF KFLHSAISTL YTRVNGP GV ADLCLRLFCS CGQVADMTEF EEVAYEFFAQ AFTVYEESIS DSKAQFQAVC VIASALHRTR NFGRENYDTL ITKCAQHA S KLLRKPDQCR AVYLASHLWW ATPIAARGET EDTELYRDGK RVLECLQRAL RVADSCMETA TSIELFVEIL DRYVYYFDQ RNESVTTKYL NGLIELIHSN LAGNQQDSAS VEASRKHFIQ TLEMIQ UniProtKB: Vacuolar protein sorting-associated protein 35 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | 3D array |
-Sample preparation
Concentration | 1.1 mg/mL | |||||||||
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Buffer | pH: 7.5 Component:
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 292 K / Instrument: LEICA EM GP | |||||||||
Details | The solution-assembled complex was incubated with Folch liposomes at room temperature. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Average electron dose: 3.17 e/Å2 Details: Tomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017) |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
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). |
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Final reconstruction | Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 11.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 16037 |
Extraction | Number tomograms: 71 / Number images used: 194885 / Reference model: reference-free Method: geometrical seeding alogn the surface of manually traced tubules |
Final angle assignment | Type: OTHER / Software: (Name: AV3, TOM) Details: Image processing was performed in TOM Toolbox, AV3 and Dynamo toolboxes. |
-Atomic model buiding 1
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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Refinement | Protocol: FLEXIBLE FIT |
Output model | PDB-6h7w: |