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Basic information

Entry
Database: EMDB / ID: EMD-2875
TitleCryo electron microscopy of actively translating human polysomes (POST state).
Map dataPOST state of human polysomes
Sample
  • Sample: Native ribosomal complex from polysomes - POST state
  • Complex: 80S ribosomal complex
Keywordsmammalian ribosome / translation / polysome / cryo electron microscopy / elongation cycle
Function / homology
Function and homology information


embryonic brain development / translation at presynapse / response to insecticide / negative regulation of endoplasmic reticulum unfolded protein response / eukaryotic 80S initiation complex / ribosomal protein import into nucleus / regulation of G1 to G0 transition / oxidized pyrimidine DNA binding / response to TNF agonist / positive regulation of base-excision repair ...embryonic brain development / translation at presynapse / response to insecticide / negative regulation of endoplasmic reticulum unfolded protein response / eukaryotic 80S initiation complex / ribosomal protein import into nucleus / regulation of G1 to G0 transition / oxidized pyrimidine DNA binding / response to TNF agonist / positive regulation of base-excision repair / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage / positive regulation of respiratory burst involved in inflammatory response / positive regulation of gastrulation / regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / protein tyrosine kinase inhibitor activity / IRE1-RACK1-PP2A complex / TNFR1-mediated ceramide production / positive regulation of Golgi to plasma membrane protein transport / G1 to G0 transition / negative regulation of formation of translation preinitiation complex / nucleolus organization / positive regulation of ubiquitin-protein transferase activity / positive regulation of DNA-templated transcription initiation / negative regulation of RNA splicing / GAIT complex / negative regulation of DNA repair / TORC2 complex binding / erythrocyte homeostasis / supercoiled DNA binding / regulation of establishment of cell polarity / rRNA modification in the nucleus and cytosol / oxidized purine DNA binding / NF-kappaB complex / negative regulation of intrinsic apoptotic signaling pathway in response to hydrogen peroxide / negative regulation of phagocytosis / ubiquitin-like protein conjugating enzyme binding / cytoplasmic translational initiation / cytoplasmic side of rough endoplasmic reticulum membrane / regulation of translation involved in cellular response to UV / A band / Formation of the ternary complex, and subsequently, the 43S complex / laminin receptor activity / ion channel inhibitor activity / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / response to aldosterone / negative regulation of myoblast fusion / protein-DNA complex disassembly / Ribosomal scanning and start codon recognition / protein kinase A binding / Translation initiation complex formation / negative regulation of Wnt signaling pathway / positive regulation of DNA damage response, signal transduction by p53 class mediator / fibroblast growth factor binding / BH3 domain binding / Protein hydroxylation / TOR signaling / mTORC1-mediated signalling / negative regulation of translational frameshifting / monocyte chemotaxis / PELO:HBS1L and ABCE1 dissociate a ribosome on a non-stop mRNA / SRC activates STAT3 in a quantitative manner, through Cadherin-11 (CDH11), RAC1 and gp130 (IL6ST) / SARS-CoV-1 modulates host translation machinery / regulation of cell division / positive regulation of GTPase activity / protein localization to nucleus / Peptide chain elongation / Selenocysteine synthesis / negative regulation of protein binding / Formation of a pool of free 40S subunits / negative regulation of respiratory burst involved in inflammatory response / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / protein targeting / protein serine/threonine kinase inhibitor activity / Eukaryotic Translation Termination / Dengue Virus Attachment and Entry / SRP-dependent cotranslational protein targeting to membrane / Response of EIF2AK4 (GCN2) to amino acid deficiency / ubiquitin ligase inhibitor activity / Viral mRNA Translation / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / positive regulation of signal transduction by p53 class mediator / GTP hydrolysis and joining of the 60S ribosomal subunit / embryo implantation / L13a-mediated translational silencing of Ceruloplasmin expression / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / Major pathway of rRNA processing in the nucleolus and cytosol / spindle assembly / positive regulation of microtubule polymerization / phagocytic cup / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / maturation of LSU-rRNA / negative regulation of ubiquitin-dependent protein catabolic process / Protein methylation / positive regulation of cell cycle / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translation regulator activity / Nuclear events stimulated by ALK signaling in cancer / DNA-(apurinic or apyrimidinic site) endonuclease activity
Similarity search - Function
60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / 40S ribosomal protein SA / 40S ribosomal protein SA, C-terminal domain / 40S ribosomal protein SA C-terminus / Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / 50S ribosomal protein L10, insertion domain superfamily / Ubiquitin-like protein FUBI ...60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / 40S ribosomal protein SA / 40S ribosomal protein SA, C-terminal domain / 40S ribosomal protein SA C-terminus / Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / 50S ribosomal protein L10, insertion domain superfamily / Ubiquitin-like protein FUBI / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / Ribosomal protein L30e / Ribosomal protein L28e / Ribosomal L15/L27a, N-terminal / : / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / Ribosomal protein L23 / Ribosomal protein L1, conserved site / Ribosomal protein L2, archaeal-type / Ribosomal protein L1 signature. / Ribosomal protein L1 / metallochaperone-like domain / TRASH domain / Ribosomal protein L1, 3-layer alpha/beta-sandwich / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / : / Ribosomal protein S12e signature. / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein S12e / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein S21e / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal L38e protein family / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / : / Ribosomal protein S2, eukaryotic / Ribosomal protein L19, eukaryotic / Ribosomal protein L6e signature. / 60S ribosomal protein L18a/ L20, eukaryotes / 40S Ribosomal protein S10 / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / S27a-like superfamily / Plectin/S10, N-terminal / Ribosomal protein L18/L18-A/B/e, conserved site / Ribosomal protein L19/L19e conserved site / Plectin/S10 domain / Ribosomal protein L19e signature. / Ribosomal protein L18e signature. / Ribosomal protein L44e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein L24e signature. / Ribosomal protein L10e / Ribosomal protein S30 / Ribosomal protein S30 / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal protein L23, N-terminal domain / Ribosomal L18 C-terminal region / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein S10, eukaryotic/archaeal / Ribosomal L40e family / Ribosomal protein S25 / Ribosomal protein S8e subdomain, eukaryotes / S25 ribosomal protein / Ribosomal protein L36e signature. / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein L30e signature 1. / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Eukaryotic Ribosomal Protein L27, KOW domain
Similarity search - Domain/homology
Large ribosomal subunit protein uL10 / Small ribosomal subunit protein eS17 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein eL33 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL22 / Small ribosomal subunit protein eS4, Y isoform 1 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein eS12 ...Large ribosomal subunit protein uL10 / Small ribosomal subunit protein eS17 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein eL33 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL22 / Small ribosomal subunit protein eS4, Y isoform 1 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein eS12 / Large ribosomal subunit protein eL13 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL22 / Large ribosomal subunit protein uL4 / Small ribosomal subunit protein eS19 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL13 / Small ribosomal subunit protein eS27 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein eL21 / Large ribosomal subunit protein eL28 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein eS10 / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL34 / Large ribosomal subunit protein eL14 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein eS1 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein eL15 / Large ribosomal subunit protein eL27 / Large ribosomal subunit protein eL43 / Large ribosomal subunit protein eL37 / Small ribosomal subunit protein eS7 / Small ribosomal subunit protein eS8 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein eL8 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein eS6 / Large ribosomal subunit protein uL14 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein eS24 / Small ribosomal subunit protein eS25 / Small ribosomal subunit protein eS26 / Small ribosomal subunit protein eS28 / Ubiquitin-like FUBI-ribosomal protein eS30 fusion protein / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein eL31 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein eL32 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL2 / Small ribosomal subunit protein eS32 / Ubiquitin-ribosomal protein eS31 fusion protein / Ubiquitin-ribosomal protein eL40 fusion protein / Large ribosomal subunit protein eL38 / Small ribosomal subunit protein eS21 / Small ribosomal subunit protein RACK1 / Large ribosomal subunit protein eL24 / Large ribosomal subunit protein eL42 / Large ribosomal subunit protein eL19 / Large ribosomal subunit protein eL20 / Large ribosomal subunit protein eL6 / Large ribosomal subunit protein eL18 / Large ribosomal subunit protein eL36
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBehrmann E / Loerke J / Budkevich TV / Yamamoto K / Schmidt A / Penczek PA / Vos MR / Burger J / Mielke T / Scheerer P / Spahn CMT
CitationJournal: 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
DepositionFeb 6, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseMay 13, 2015-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

post.tif

Downloads & links

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Map

FileDownload / File: emd_2875.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPOST state of human polysomes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 400 pix.
= 378. Å		0.95 Å/pix.
x 400 pix.
= 378. Å		0.95 Å/pix.
x 400 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.945 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-7.9781189 - 15.02807808
Average (Standard dev.)0.00000404 (±1.00000489)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9450.9450.945
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-7.97815.0280.000

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Supplemental data

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Sample components

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Entire : Native ribosomal complex from polysomes - POST state

EntireName: Native ribosomal complex from polysomes - POST state
Components
  • Sample: Native ribosomal complex from polysomes - POST state
  • Complex: 80S ribosomal complex

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Supramolecule #1000: Native ribosomal complex from polysomes - POST state

SupramoleculeName: Native ribosomal complex from polysomes - POST state / type: sample / ID: 1000 / Details: The sample was monodisperse / Number unique components: 1
Molecular weightExperimental: 4.5 MDa / Theoretical: 4.5 MDa

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Supramolecule #1: 80S ribosomal complex

SupramoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / Strain: HEK 293T / synonym: Human / Tissue: Kidney / Cell: HEK 293T / Location in cell: Cytoplasm
Molecular weightExperimental: 4.5 MDa / Theoretical: 4.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.5 mg/mL
BufferpH: 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
GridDetails: Quantifoil grids with additional continuous carbon support
VitrificationCryogen 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 ID1
MicroscopeFEI POLARA 300
DetailsData was collected automatically with Leginon
DateAug 20, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 51282 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TITAN KRIOS
DateNov 29, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 51282 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied 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

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