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Yorodumi- EMDB-11607: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with... -
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-Basic information
Entry | Database: EMDB / ID: EMD-11607 | ||||||||||||
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Title | Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer | ||||||||||||
Map data | |||||||||||||
Sample |
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Function / homology | Function and homology information GET complex / pheromone-dependent signal transduction involved in conjugation with cellular fusion / tail-anchored membrane protein insertion into ER membrane / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / response to arsenic-containing substance / response to metal ion / mitophagy ...GET complex / pheromone-dependent signal transduction involved in conjugation with cellular fusion / tail-anchored membrane protein insertion into ER membrane / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / response to arsenic-containing substance / response to metal ion / mitophagy / protein-membrane adaptor activity / protein folding chaperone / guanyl-nucleotide exchange factor activity / mitochondrial membrane / unfolded protein binding / response to heat / Golgi membrane / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / metal ion binding / cytosol Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 14.0 Å | ||||||||||||
Authors | McDowell MA / Pfeffer S / Flemming D / Sinning I | ||||||||||||
Funding support | Germany, 3 items
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Citation | Journal: Mol Cell / Year: 2020 Title: Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex. Authors: Melanie A McDowell / Michael Heimes / Francesco Fiorentino / Shahid Mehmood / Ákos Farkas / Javier Coy-Vergara / Di Wu / Jani Reddy Bolla / Volker Schmid / Roger Heinze / Klemens Wild / ...Authors: Melanie A McDowell / Michael Heimes / Francesco Fiorentino / Shahid Mehmood / Ákos Farkas / Javier Coy-Vergara / Di Wu / Jani Reddy Bolla / Volker Schmid / Roger Heinze / Klemens Wild / Dirk Flemming / Stefan Pfeffer / Blanche Schwappach / Carol V Robinson / Irmgard Sinning / Abstract: Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and ...Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion. | ||||||||||||
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_11607.map.gz | 2.8 MB | EMDB map data format | |
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Header (meta data) | emd-11607-v30.xml emd-11607.xml | 15.9 KB 15.9 KB | Display Display | EMDB header |
Images | emd_11607.png | 32.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-11607 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-11607 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_11607.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 2.14 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 : Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with...
Entire | Name: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer |
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Components |
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-Supramolecule #1: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with...
Supramolecule | Name: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Molecular weight | Experimental: 174 KDa |
-Supramolecule #2: ATPase Get3
Supramolecule | Name: ATPase Get3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #3: Get1 and Get2
Supramolecule | Name: Get1 and Get2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) |
-Macromolecule #1: Get1
Macromolecule | Name: Get1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Sequence | String: MHWAAAVAIF FIVVTKFLQY TNK YHEKWI SKFAPGNELS KKYLAKVKER HELKEFNNSI SAQDNYAKWT KNNRKLDSLD KEIN NLKDE IQSENKAFQA HLHKLRLLAL TVPFFVFKIM YGKTPVYKLS SSTSTLFPTF VSGVW SQGW LYVLLHPLRT ISQKWHIMEG ...String: MHWAAAVAIF FIVVTKFLQY TNK YHEKWI SKFAPGNELS KKYLAKVKER HELKEFNNSI SAQDNYAKWT KNNRKLDSLD KEIN NLKDE IQSENKAFQA HLHKLRLLAL TVPFFVFKIM YGKTPVYKLS SSTSTLFPTF VSGVW SQGW LYVLLHPLRT ISQKWHIMEG KFGASKFDDM ALQSVSLGIW VWALMNVING VEFIVK QLF LTPKMEAPAS VETQEEKA L DAVDDAIILD GSLEVLFQ |
-Macromolecule #2: Get2
Macromolecule | Name: Get2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Sequence | String: MDYHDYLLNR LKAWTILVKW VFFLLPYLYL ITRPN SSVW PAYAFTQSAW FAPLRNPSNF TRIFATFEFL SISIYYQLLK NVEHKSKIKN LQDTNK LVK LVSLVPEGVI PVANLKGKLI T LLQYWD L LSMLITDISF VLIVLGLLTY LGSGSGSGSG SGSGS |
-Macromolecule #3: Get3
Macromolecule | Name: Get3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MDLTVEPNLH SLITSTTHK W IFVGGKGG VG KTTSSCS IAI QMALSQ PNKQ FLLIS TDPAH NLSD AFGEKF GKD ARKVTGM NN LSCMEIDP S AALKDMNDM AVSRANNNGS DGQGDDLGS L LQGGALAD LT GSIPGID EAL SFMEVM KHIK RQEQG ...String: MDLTVEPNLH SLITSTTHK W IFVGGKGG VG KTTSSCS IAI QMALSQ PNKQ FLLIS TDPAH NLSD AFGEKF GKD ARKVTGM NN LSCMEIDP S AALKDMNDM AVSRANNNGS DGQGDDLGS L LQGGALAD LT GSIPGID EAL SFMEVM KHIK RQEQG EGETF DTVI FDTAPT GHT LRFLQLP NT LSKLLEKF G EITNKLGPM LNSFMGAGNV DISGKLNEL K ANVETIRQ QF TDPDLTT FVC VCISEF LSLY ETERL IQELI SYDM DVNSII VNQ LLFAEND QE HNCKRCQA R WKMQKKYLD QIDELYEDFH VVKMPLCAG E IRGLNNLT KF SQFLNKE YNP ITDGKV IYEL EDKEW SHPQF EK |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.3 mg/mL | ||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE | ||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV | ||||||
Details | complex stabilised in A835 amphipol |
-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: 2.5 µm / Nominal defocus min: 1.5 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 678 / Average exposure time: 4.0 sec. / Average electron dose: 25.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 150857 |
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CTF correction | Software - Name: Gctf |
Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.2) / Number images used: 27666 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Refinement | Protocol: RIGID BODY FIT |