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Yorodumi- EMDB-2875: Cryo electron microscopy of actively translating human polysomes ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2875 | |||||||||
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Title | Cryo electron microscopy of actively translating human polysomes (POST state). | |||||||||
Map data | POST state of human polysomes | |||||||||
Sample |
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Keywords | mammalian ribosome / translation / polysome / cryo electron microscopy / elongation cycle | |||||||||
Function / homology | Function and homology information eukaryotic 80S initiation complex / : / negative regulation of protein neddylation / translation at presynapse / negative regulation of endoplasmic reticulum unfolded protein response / oxidized pyrimidine DNA binding / response to TNF agonist / positive regulation of base-excision repair / axial mesoderm development / ribosomal protein import into nucleus ...eukaryotic 80S initiation complex / : / negative regulation of protein neddylation / translation at presynapse / negative regulation of endoplasmic reticulum unfolded protein response / oxidized pyrimidine DNA binding / response to TNF agonist / positive regulation of base-excision repair / axial mesoderm development / ribosomal protein import into nucleus / embryonic brain development / protein tyrosine kinase inhibitor activity / positive regulation of respiratory burst involved in inflammatory response / negative regulation of formation of translation preinitiation complex / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage / positive regulation of gastrulation / nucleolus organization / regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / 90S preribosome assembly / IRE1-RACK1-PP2A complex / positive regulation of endodeoxyribonuclease activity / positive regulation of Golgi to plasma membrane protein transport / TNFR1-mediated ceramide production / negative regulation of DNA repair / negative regulation of RNA splicing / GAIT complex / negative regulation of intrinsic apoptotic signaling pathway in response to hydrogen peroxide / supercoiled DNA binding / oxidized purine DNA binding / neural crest cell differentiation / NF-kappaB complex / middle ear morphogenesis / ubiquitin-like protein conjugating enzyme binding / negative regulation of phagocytosis / regulation of establishment of cell polarity / positive regulation of ubiquitin-protein transferase activity / A band / rRNA modification in the nucleus and cytosol / erythrocyte homeostasis / Formation of the ternary complex, and subsequently, the 43S complex / alpha-beta T cell differentiation / cytoplasmic side of rough endoplasmic reticulum membrane / regulation of G1 to G0 transition / exit from mitosis / laminin receptor activity / pigmentation / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / regulation of translation involved in cellular response to UV / protein-DNA complex disassembly / positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / protein kinase A binding / negative regulation of ubiquitin protein ligase activity / optic nerve development / Ribosomal scanning and start codon recognition / ion channel inhibitor activity / response to aldosterone / Translation initiation complex formation / retinal ganglion cell axon guidance / fibroblast growth factor binding / mammalian oogenesis stage / homeostatic process / positive regulation of mitochondrial depolarization / G1 to G0 transition / activation-induced cell death of T cells / positive regulation of T cell receptor signaling pathway / macrophage chemotaxis / negative regulation of Wnt signaling pathway / lung morphogenesis / negative regulation of peptidyl-serine phosphorylation / iron-sulfur cluster binding / positive regulation of activated T cell proliferation / monocyte chemotaxis / Protein hydroxylation / regulation of cell division / BH3 domain binding / cysteine-type endopeptidase activator activity involved in apoptotic process / mTORC1-mediated signalling / SARS-CoV-1 modulates host translation machinery / Peptide chain elongation / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / Selenocysteine synthesis / positive regulation of signal transduction by p53 class mediator / Formation of a pool of free 40S subunits / blastocyst development / ubiquitin ligase inhibitor activity / Eukaryotic Translation Termination / phagocytic cup / negative regulation of respiratory burst involved in inflammatory response / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / Viral mRNA Translation / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / protein localization to nucleus / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / TOR signaling / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / T cell proliferation involved in immune response / positive regulation of cell cycle Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | Behrmann E / Loerke J / Budkevich TV / Yamamoto K / Schmidt A / Penczek PA / Vos MR / Burger J / Mielke T / Scheerer P / Spahn CMT | |||||||||
Citation | Journal: Cell / Year: 2015 Title: Structural snapshots of actively translating human ribosomes. Authors: Elmar Behrmann / Justus Loerke / Tatyana V Budkevich / Kaori Yamamoto / Andrea Schmidt / Pawel A Penczek / Matthijn R Vos / Jörg Bürger / Thorsten Mielke / Patrick Scheerer / Christian M T Spahn / Abstract: Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical ...Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements. | |||||||||
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_2875.map.gz | 152.8 MB | EMDB map data format | |
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Header (meta data) | emd-2875-v30.xml emd-2875.xml | 10.9 KB 10.9 KB | Display Display | EMDB header |
Images | post.tif | 3.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2875 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2875 | HTTPS FTP |
-Validation report
Summary document | emd_2875_validation.pdf.gz | 290.9 KB | Display | EMDB validaton report |
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Full document | emd_2875_full_validation.pdf.gz | 290.1 KB | Display | |
Data in XML | emd_2875_validation.xml.gz | 7.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2875 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2875 | HTTPS FTP |
-Related structure data
Related structure data | 5aj0MUC 2902C 2903C 2904C 2905C 2906C 2907C 2908C 2909C 2910C 2911C M: atomic model generated by this map U: unfit; in different coordinate system*YM 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_2875.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | POST state of human polysomes | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.945 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 : Native ribosomal complex from polysomes - POST state
Entire | Name: Native ribosomal complex from polysomes - POST state |
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Components |
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-Supramolecule #1000: Native ribosomal complex from polysomes - POST state
Supramolecule | Name: Native ribosomal complex from polysomes - POST state / type: sample / ID: 1000 / Details: The sample was monodisperse / Number unique components: 1 |
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Molecular weight | Experimental: 4.5 MDa / Theoretical: 4.5 MDa |
-Supramolecule #1: 80S ribosomal complex
Supramolecule | Name: 80S ribosomal complex / type: complex / ID: 1 / Name.synonym: 80S ribosome Details: The sample was isolated as polysomes from the cell. Structural analysis shows that the complex contains 2 tRNAs and mRNA. Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL |
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Source (natural) | Organism: Homo sapiens (human) / Strain: HEK 293T / synonym: Human / Tissue: Kidney / Cell: HEK 293T / Location in cell: Cytoplasm |
Molecular weight | Experimental: 4.5 MDa / Theoretical: 4.5 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3.5 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM Hepes-KOH, pH 7.5, 100 mM KCl, 1.5 mM MgCl2, 0.5 mM spermidine, 0.04 mM spermine, 1 mM DTT |
Grid | Details: Quantifoil grids with additional continuous carbon support |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK II / Method: blot for 2-4 seconds before plunging |
-Electron microscopy #1
Microscopy ID | 1 |
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Microscope | FEI POLARA 300 |
Details | Data was collected automatically with Leginon |
Date | Aug 20, 2012 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 51282 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 205000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 115000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Electron microscopy #2
Microscopy ID | 2 |
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Microscope | FEI TITAN KRIOS |
Date | Nov 29, 2012 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 51282 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 172000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 96000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each micrograph |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: OTHER / Software - Name: Signature, CTFFind3, Spider, SPARX Details: Maps were calculated from 2 datasets using SSNR-weighted combination. Number images used: 313321 |