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- PDB-8at6: Cryo-EM structure of yeast Elp456 subcomplex -

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Basic information

Entry
Database: PDB / ID: 8at6
TitleCryo-EM structure of yeast Elp456 subcomplex
Components
  • Elongator complex protein 4
  • Elongator complex protein 5
  • Elongator complex protein 6
KeywordsTRANSLATION / wobble uridine modification
Function / homology
Function and homology information


elongator holoenzyme complex / protein urmylation / tRNA wobble uridine modification / tRNA modification / transcription elongation factor complex / regulation of translation / tRNA binding / regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding ...elongator holoenzyme complex / protein urmylation / tRNA wobble uridine modification / tRNA modification / transcription elongation factor complex / regulation of translation / tRNA binding / regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Elongator complex protein 4 / Elongator complex protein 6 / Elongator complex protein 5 / PAXNEB protein / Elongator subunit Iki1 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongator complex protein 5 / Elongator complex protein 4 / Elongator complex protein 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsJaciuk, 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
OrganizationGrant numberCountry
Polish National Science Centre2018/31/B/NZ1/03559 Poland
European Research Council (ERC)101001394European Union
CitationJournal: 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
DepositionAug 22, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Elongator complex protein 4
B: Elongator complex protein 5
C: Elongator complex protein 6
D: Elongator complex protein 4
E: Elongator complex protein 5
F: Elongator complex protein 6


Theoretical massNumber of molelcules
Total (without water)234,1756
Polymers234,1756
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Complex composition and stoichiometry validated using SDS-PAGE
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16000 Å2
ΔGint-78 kcal/mol
Surface area67430 Å2
MethodPISA

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Components

#1: Protein Elongator complex protein 4 / Gamma-toxin target 7 / HAT-associated protein 1


Mass: 51232.469 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: ELP4, HAP1, TOT7, YPL101W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02884
#2: Protein Elongator complex protein 5 / / Gamma-toxin target 5 / HAT-associated protein 2 / Protein IKI1


Mass: 35252.496 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: IKI1, ELP5, HAP2, TOT5, YHR187W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38874
#3: Protein Elongator complex protein 6 / Gamma-toxin target 6 / HAT-associated protein 3


Mass: 30602.611 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: ELP6, HAP3, TOT6, YMR312W, YM9924.04 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04868

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Yeast Elp456 subcomplex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2338 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
220 mMHEPESHEPES1
33 mMDithiothreitolDithiothreitol1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.82 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 4716
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
2EPU2.1image acquisition
4cryoSPARCv3.2.0CTF correctionpatch CTF
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCv3.2.0initial Euler assignmentAb-Initio Reconstruction
11RELION3.1final Euler assignment3D auto-refine
12RELION3.1classificationmasked 3D classification
13RELION3.13D reconstructionPostprocessing
Image processingDetails: 20 eV slit, fully tuned before the experiment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 364424
Details: given number of particles is after TOPAZ picking and 2D cleaning
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128593
Details: Given global resolution estimation for post-process sharpened map
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: After initial rigid body fit, further fitting was done using NAMDINATOR
Atomic model buildingPDB-ID: 4A8J

4a8j

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