Cryo-EM reconstruction of the HeLa large ribosomal subunit.
試料
複合体: Human mature large ribosomal subunit
タンパク質・ペプチド: x 41種
RNA: x 3種
リガンド: x 8種
キーワード
Large ribosomal subunit / HeLa / modifications / polyamines / RIBOSOME
機能・相同性
機能・相同性情報
eukaryotic 80S initiation complex / negative regulation of protein neddylation / regulation of G1 to G0 transition / axial mesoderm development / negative regulation of formation of translation preinitiation complex / ribosomal protein import into nucleus / 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 / 90S preribosome assembly ...eukaryotic 80S initiation complex / negative regulation of protein neddylation / regulation of G1 to G0 transition / axial mesoderm development / negative regulation of formation of translation preinitiation complex / ribosomal protein import into nucleus / 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 / 90S preribosome assembly / GAIT complex / A band / positive regulation of DNA damage response, signal transduction by p53 class mediator / TORC2 complex binding / alpha-beta T cell differentiation / G1 to G0 transition / middle ear morphogenesis / exit from mitosis / translation at presynapse / optic nerve development / cytoplasmic side of rough endoplasmic reticulum membrane / retinal ganglion cell axon guidance / negative regulation of ubiquitin protein ligase activity / homeostatic process / response to aldosterone / macrophage chemotaxis / lung morphogenesis / male meiosis I / Protein hydroxylation / Peptide chain elongation / Selenocysteine synthesis / positive regulation of signal transduction by p53 class mediator / Formation of a pool of free 40S subunits / ubiquitin ligase inhibitor activity / Eukaryotic Translation Termination / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / blastocyst development / negative regulation of ubiquitin-dependent protein catabolic process / cellular response to actinomycin D / Viral mRNA Translation / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / protein localization to nucleus / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / Major pathway of rRNA processing in the nucleolus and cytosol / protein targeting / protein-RNA complex assembly / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / maturation of LSU-rRNA / rough endoplasmic reticulum / Maturation of protein E / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / Maturation of protein E / MDM2/MDM4 family protein binding / cytosolic ribosome / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / embryo implantation / Membrane binding and targetting of GAG proteins / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Regulation of FZD by ubiquitination / Downregulation of ERBB4 signaling / APC-Cdc20 mediated degradation of Nek2A / p75NTR recruits signalling complexes / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / Regulation of innate immune responses to cytosolic DNA / NF-kB is activated and signals survival / cellular response to interleukin-4 / Downregulation of ERBB2:ERBB3 signaling / ossification / Pexophagy / NRIF signals cell death from the nucleus / innate immune response in mucosa / VLDLR internalisation and degradation / Regulation of PTEN localization / DNA damage response, signal transduction by p53 class mediator / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Regulation of BACH1 activity 類似検索 - 分子機能
Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / Ribosomal protein L30e / Ribosomal protein L2, archaeal-type / Ribosomal L15/L27a, N-terminal / Ribosomal protein L28e / Ribosomal protein L23 / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / metallochaperone-like domain ...Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / Ribosomal protein L30e / Ribosomal protein L2, archaeal-type / Ribosomal L15/L27a, N-terminal / Ribosomal protein L28e / Ribosomal protein L23 / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / metallochaperone-like domain / TRASH domain / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L10e / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / Ribosomal protein L44e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein L24e signature. / : / Ribosomal protein L19, eukaryotic / Ribosomal protein L19/L19e conserved site / Ribosomal protein L19e signature. / Ribosomal protein L6e signature. / Ribosomal protein L13e / Ribosomal protein L13e / 60S ribosomal protein L18a/ L20, eukaryotes / : / Ribosomal protein L44e / Ribosomal protein L44 / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L30e signature 1. / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal L40e family / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Ribosomal protein L23/L25, N-terminal / : / Ribosomal protein L23, N-terminal domain / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein 60S L18 and 50S L18e / Ribosomal Protein L6, KOW domain / Ribosomal protein L18/L18-A/B/e, conserved site / Ribosomal protein L18e signature. / Ribosomal protein L30e signature 2. / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal L27e protein family / Ribosomal protein L36e signature. / Ribosomal protein L35Ae, conserved site / Ribosomal protein L30e, conserved site / Ribosomal protein L35Ae signature. / Ribosomal protein L39e, conserved site / Ribosomal protein L39e signature. / : / Ribosomal protein L6e / Ribosomal protein L34Ae / Ribosomal protein L34e / 60S ribosomal protein L19 / Ribosomal protein L30/YlxQ / Ribosomal protein L7A/L8 / 60S ribosomal protein L6E / 60S ribosomal protein L35 / Ribosomal protein L13, eukaryotic/archaeal / Ribosomal protein L18e / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L4 C-terminal domain / Ribosomal protein L7, eukaryotic / Ribosomal protein L30, N-terminal / Ribosomal protein L31e, conserved site / Ribosomal L30 N-terminal domain / Ribosomal protein L31e signature. / Ribosomal protein L37ae / Ribosomal L37ae protein family / Ribosomal protein L36e / Ribosomal protein L36e domain superfamily / Ribosomal protein L36e / Ribosomal_L19e / Ribosomal protein L19/L19e / Ribosomal protein L19/L19e, domain 1 / Ribosomal protein L19/L19e superfamily / Ribosomal protein L19e, N-terminal domain / Ribosomal protein L14e domain / Ribosomal protein L14 類似検索 - ドメイン・相同性
Large ribosomal subunit protein eL33 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein eL13 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL22 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL29 ...Large ribosomal subunit protein eL33 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein eL13 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL22 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL13 / 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 / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL34 / Large ribosomal subunit protein eL14 / 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 / Large ribosomal subunit protein eL8 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein eL31 / Large ribosomal subunit protein eL32 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL2 / Ubiquitin-ribosomal protein eL40 fusion protein / Large ribosomal subunit protein eL38 / 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 / Ribosomal protein uL16-like / Large ribosomal subunit protein eL36 類似検索 - 構成要素
ジャーナル: Nucleic Acids Res / 年: 2025 タイトル: Visualizing the modification landscape of the human 60S ribosomal subunit at close to atomic resolution. 著者: Franziska Wiechert / Anett Unbehaun / Thiemo Sprink / Helena Seibel / Jörg Bürger / Justus Loerke / Thorsten Mielke / Christoph A Diebolder / Magdalena Schacherl / Christian M T Spahn / 要旨: Chemical modifications of ribosomal RNAs (rRNAs) and proteins expand their topological repertoire, and together with the plethora of bound ligands, fine-tune ribosomal function. Detailed knowledge of ...Chemical modifications of ribosomal RNAs (rRNAs) and proteins expand their topological repertoire, and together with the plethora of bound ligands, fine-tune ribosomal function. Detailed knowledge of this natural composition provides important insights into ribosome genesis and function and clarifies some aspects of ribosomopathies. The discovery of new structural properties and functional aspects of ribosomes has gone hand in hand with cryo-electron microscopy (cryo-EM) and its technological development. In line with the ability to visualize atomic details - a prerequisite for identifying chemical modifications and ligands in cryo-EM maps - in this work we present the structure of the 60S ribosomal subunit from HeLa cells at the very high global resolution of 1.78 Å. We identified 113 rRNA modifications and four protein modifications including uL2-Hisβ-ox216, which stabilizes the local structure near the peptidyl transferase centre via an extended hydrogen-bonding network. We can differentiate metal ions Mg2+ and K+, polyamines spermine, spermidine and putrescine and identify thousands of water molecules binding to the 60S subunit. Approaching atomic resolution cryo-EM has become a powerful tool to examine fine details of macromolecular structures that will expand our knowledge about translation and other biological processes in the future and assess the variability of the chemical space due to differences between species/tissues or varying physicochemical environment.
凍結剤: ETHANE / チャンバー内湿度: 85 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV
詳細
Hela 60S ribosomal subunits were isolated from cytoplasmic extracts of Hela cells, involving puromycin treatment and sucrose gradient centrifugation in high salt. The gradients were manually fractionated, and separated peak fractions of 60S subunits were collected. For buffer exchange, the sample was diluted 6 x in buffer F (20 mM HEPES pH7.5, 10 mM KCl, 2 mM MgCl2, 2 mM DTT) supplemented with 8.56 % sucrose to adjust the sucrose in the storage buffer to 12%, respectively. Finally, the 60S subunit suspension was concentrated on Amicon Ultracel filter units with a 10 kDa cutoff (Merck Millipore) was snap frozen in 10 ul aliquots and stored at -80 degrees Celsius. Concentrated Hela 60S ribosomal subunits were diluted to 200 nM in buffer E (20 mM HEPES pH7.5, 2 mM MgCl2, 90 mM KCl, 0.5 mM Spermidine, 0.04 mM Spermine, 2 mM DTT) and 4 ul were spotted onto glow-discharged holey carbon grids coated with a continuous thin carbon film (Quantifoil Cu200 R2/2+) and incubated for 40 sec at 4 degrees CelsiusC, 85% RH. The excess of liquid was blotted away for 2 sec and the grids were plunge frozen using the Vitrobot Mark IV (Thermo Fischer Scientific).
Chain - Source name: PDB / Chain - Initial model type: experimental model
詳細
Interactive model building was performed in Coot using an auto sharpened map in cryoSPARC and additionally a low-pass-filtered map to 4 Angstrom for the more mobile regions of the 60S structure. Each residue of the model was checked individually for modifications and adjusted manually with a subsequent refinement in phenix.refine. Polyamines and metals were added while searching for unmodelled blobs in Coot. Water molecules were added using phenix.douse and visually validated in Coot. The model was validated using Molprobity.
精密化
空間: REAL / プロトコル: OTHER 当てはまり具合の基準: Cross-correlation coefficient
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試料
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機能・相同性情報
Homo sapiens (ヒト)
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ドイツ, 1件 
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emd_18765.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-18765
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