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- EMDB-2875: Cryo electron microscopy of actively translating human polysomes ... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2875 | |||||||||
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Title | Cryo electron microscopy of actively translating human polysomes (POST state). | |||||||||
![]() | POST state of human polysomes | |||||||||
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![]() | mammalian ribosome / translation / polysome / cryo electron microscopy / elongation cycle | |||||||||
Function / homology | ![]() positive regulation of cysteine-type endopeptidase activity involved in execution phase of apoptosis / negative regulation of endoplasmic reticulum unfolded protein response / embryonic brain development / oxidized pyrimidine DNA binding / eukaryotic 80S initiation complex / response to TNF agonist / positive regulation of base-excision repair / negative regulation of protein neddylation / protein tyrosine kinase inhibitor activity / positive regulation of respiratory burst involved in inflammatory response ...positive regulation of cysteine-type endopeptidase activity involved in execution phase of apoptosis / negative regulation of endoplasmic reticulum unfolded protein response / embryonic brain development / oxidized pyrimidine DNA binding / eukaryotic 80S initiation complex / response to TNF agonist / positive regulation of base-excision repair / negative regulation of protein neddylation / protein tyrosine kinase inhibitor activity / positive regulation of respiratory burst involved in inflammatory response / translation at presynapse / regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage / positive regulation of gastrulation / axial mesoderm development / negative regulation of formation of translation preinitiation complex / nucleolus organization / ribosomal protein import into nucleus / IRE1-RACK1-PP2A complex / : / positive regulation of endodeoxyribonuclease activity / positive regulation of Golgi to plasma membrane protein transport / 90S preribosome assembly / TNFR1-mediated ceramide production / negative regulation of RNA splicing / negative regulation of DNA repair / TORC2 complex binding / GAIT complex / negative regulation of intrinsic apoptotic signaling pathway in response to hydrogen peroxide / oxidized purine DNA binding / supercoiled DNA binding / neural crest cell differentiation / NF-kappaB complex / middle ear morphogenesis / ubiquitin-like protein conjugating enzyme binding / regulation of establishment of cell polarity / negative regulation of phagocytosis / positive regulation of ubiquitin-protein transferase activity / Formation of the ternary complex, and subsequently, the 43S complex / rRNA modification in the nucleus and cytosol / erythrocyte homeostasis / cytoplasmic side of rough endoplasmic reticulum membrane / A band / laminin receptor activity / regulation of G1 to G0 transition / exit from mitosis / alpha-beta T cell differentiation / 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 kinase A binding / protein-DNA complex disassembly / positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / Translation initiation complex formation / Ribosomal scanning and start codon recognition / negative regulation of ubiquitin protein ligase activity / optic nerve development / ion channel inhibitor activity / pigmentation / mammalian oogenesis stage / positive regulation of mitochondrial depolarization / response to aldosterone / retinal ganglion cell axon guidance / G1 to G0 transition / homeostatic process / activation-induced cell death of T cells / lung morphogenesis / negative regulation of Wnt signaling pathway / fibroblast growth factor binding / positive regulation of T cell receptor signaling pathway / positive regulation of activated T cell proliferation / iron-sulfur cluster binding / regulation of cell division / Protein hydroxylation / negative regulation of peptidyl-serine phosphorylation / BH3 domain binding / mTORC1-mediated signalling / macrophage chemotaxis / SARS-CoV-1 modulates host translation machinery / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / Peptide chain elongation / monocyte chemotaxis / Selenocysteine synthesis / cysteine-type endopeptidase activator activity involved in apoptotic process / positive regulation of signal transduction by p53 class mediator / ubiquitin ligase inhibitor activity / Formation of a pool of free 40S subunits / phagocytic cup / Eukaryotic Translation Termination / blastocyst development / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / negative regulation of respiratory burst involved in inflammatory response / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Viral mRNA Translation / protein localization to nucleus / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / L13a-mediated translational silencing of Ceruloplasmin expression / GTP hydrolysis and joining of the 60S ribosomal subunit / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / TOR signaling Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
![]() | Behrmann E / Loerke J / Budkevich TV / Yamamoto K / Schmidt A / Penczek PA / Vos MR / Burger J / Mielke T / Scheerer P / Spahn CMT | |||||||||
![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 152.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.9 KB 10.9 KB | Display Display | ![]() |
Images | ![]() | 3.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 290.9 KB | Display | ![]() |
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Full document | ![]() | 290.1 KB | Display | |
Data in XML | ![]() | 7.5 KB | Display | |
Arichive directory | ![]() ![]() | 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 ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | POST state of human polysomes | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
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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: ![]() |
Molecular weight | Experimental: 4.5 MDa / Theoretical: 4.5 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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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 |
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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: ![]() |
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 |
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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: ![]() |
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 |
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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 |