+Open data
-Basic information
Entry | Database: PDB / ID: 8asv | |||||||||
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Title | Cryo-EM structure of yeast Elongator complex | |||||||||
Components | (Elongator complex protein ...) x 6 | |||||||||
Keywords | TRANSLATION / wobble uridine modification | |||||||||
Function / homology | Function and homology information tRNA carboxymethyluridine synthase / tRNA uridine(34) acetyltransferase activity / elongator holoenzyme complex / tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation / protein urmylation / tRNA wobble uridine modification / tRNA modification / transcription elongation factor complex / : / protein transport ...tRNA carboxymethyluridine synthase / tRNA uridine(34) acetyltransferase activity / elongator holoenzyme complex / tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation / protein urmylation / tRNA wobble uridine modification / tRNA modification / transcription elongation factor complex / : / protein transport / regulation of translation / 4 iron, 4 sulfur cluster binding / microtubule binding / tRNA binding / regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.35 Å | |||||||||
Authors | Jaciuk, M. / Scherf, D. / Kaszuba, K. / Gaik, M. / Koscielniak, A. / Krutyholowa, R. / Rawski, M. / Indyka, P. / Biela, A. / Dobosz, D. ...Jaciuk, M. / Scherf, D. / Kaszuba, K. / Gaik, M. / Koscielniak, A. / Krutyholowa, R. / Rawski, M. / Indyka, P. / Biela, A. / Dobosz, D. / Lin, T.-Y. / Abbassi, N. / Hammermeister, A. / Chramiec-Glabik, A. / Kosinski, J. / Schaffrath, R. / Glatt, S. | |||||||||
Funding support | Poland, European Union, 2items
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Citation | Journal: Nucleic Acids Res / Year: 2023 Title: Cryo-EM structure of the fully assembled Elongator complex. Authors: Marcin Jaciuk / David Scherf / Karol Kaszuba / Monika Gaik / Alexander Rau / Anna Kościelniak / Rościsław Krutyhołowa / Michał Rawski / Paulina Indyka / Andrea Graziadei / Andrzej ...Authors: Marcin Jaciuk / David Scherf / Karol Kaszuba / Monika Gaik / Alexander Rau / Anna Kościelniak / Rościsław Krutyhołowa / Michał Rawski / Paulina Indyka / Andrea Graziadei / Andrzej Chramiec-Głąbik / Anna Biela / Dominika Dobosz / Ting-Yu Lin / Nour-El-Hana Abbassi / Alexander Hammermeister / Juri Rappsilber / Jan Kosinski / Raffael Schaffrath / Sebastian Glatt / Abstract: Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition ...Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8asv.cif.gz | 809.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8asv.ent.gz | 649.3 KB | Display | PDB format |
PDBx/mmJSON format | 8asv.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8asv_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8asv_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 8asv_validation.xml.gz | 133.9 KB | Display | |
Data in CIF | 8asv_validation.cif.gz | 200.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/as/8asv ftp://data.pdbj.org/pub/pdb/validation_reports/as/8asv | HTTPS FTP |
-Related structure data
Related structure data | 15622MC 8aswC 8at6C 8avgC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Elongator complex protein ... , 6 types, 10 molecules ADBCEHFIGJ
#1: Protein | Mass: 153166.266 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: IKI3, ELP1, TOT1, YLR384C, L3502.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q06706 #2: Protein | | Mass: 89519.430 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: ELP2, TOT2, YGR200C, G7725 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P42935 #3: Protein | | Mass: 63755.059 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: ELP3, HPA1, TOT3, YPL086C / Production host: Saccharomyces cerevisiae (brewer's yeast) References: UniProt: Q02908, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups #4: Protein | Mass: 35252.496 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: IKI1, ELP5, HAP2, TOT5, YHR187W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38874 #5: Protein | Mass: 30602.611 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: ELP6, HAP3, TOT6, YMR312W, YM9924.04 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04868 #6: Protein | Mass: 51232.469 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: ELP4, HAP1, TOT7, YPL101W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02884 |
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-Non-polymers , 1 types, 1 molecules
#7: Chemical | ChemComp-SF4 / |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Yeast Elongator complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.8458 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 15 s wait time, blot force 5, 5 s blot time |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.82 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 6339 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 5760 / Height: 4092 |
-Processing
EM software |
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Image processing | Details: 20 eV slit, fully tuned before the experiment | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 188389 Details: given number of particles is after TOPAZ picking and 2D cleaning | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12514 / Details: 3D auto-refine followed by Post-processing / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT Details: After initial rigid body fit, further fitting was done using MDFF and NAMDINATOR | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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