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- PDB-8avg: Cryo-EM structure of mouse Elp123 with bound SAM -

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Basic information

Entry
Database: PDB / ID: 8avg
TitleCryo-EM structure of mouse Elp123 with bound SAM
Components
  • Elongator complex protein 1
  • Elongator complex protein 2
  • Elongator complex protein 3
KeywordsTRANSLATION / wobble uridine modification
Function / homology
Function and homology information


phosphorylase kinase regulator activity / tRNA uridine(34) acetyltransferase activity / tRNA carboxymethyluridine synthase / elongator holoenzyme complex / tRNA wobble uridine modification / regulation of receptor signaling pathway via JAK-STAT / RNA polymerase II complex binding / endopeptidase activator activity / proteasome assembly / proteasome complex ...phosphorylase kinase regulator activity / tRNA uridine(34) acetyltransferase activity / tRNA carboxymethyluridine synthase / elongator holoenzyme complex / tRNA wobble uridine modification / regulation of receptor signaling pathway via JAK-STAT / RNA polymerase II complex binding / endopeptidase activator activity / proteasome assembly / proteasome complex / central nervous system development / transcription elongation factor complex / transcription elongation by RNA polymerase II / neuron migration / 4 iron, 4 sulfur cluster binding / tRNA binding / positive regulation of cell migration / regulation of transcription by RNA polymerase II / protein kinase binding / nucleolus / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Elongator complex protein 1 / Elongator complex protein 2 / : / : / : / : / : / ELP1 first N-terminal beta-propeller / ELP1 N-terminal second beta-propeller / ELP1 TPR domain ...Elongator complex protein 1 / Elongator complex protein 2 / : / : / : / : / : / ELP1 first N-terminal beta-propeller / ELP1 N-terminal second beta-propeller / ELP1 TPR domain / ELP1 alpha-solenoid / ELP1 three-helix bundle / : / ELP3 N-terminal domain / Radical SAM, C-terminal extension / Elongator complex protein 3-like / ELP3/YhcC / Radical_SAM C-terminal domain / Elp3/MiaB/NifB / Elongator protein 3, MiaB family, Radical SAM / Radical SAM superfamily / Radical SAM core domain profile. / Radical SAM / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain / Acyl-CoA N-acyltransferase / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / IRON/SULFUR CLUSTER / Elongator complex protein 1 / Elongator complex protein 2 / Elongator complex protein 3
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.01 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 26, 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
Revision 1.3Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Elongator complex protein 1
B: Elongator complex protein 2
C: Elongator complex protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)306,1825
Polymers305,4323
Non-polymers7502
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Complex composition and stoichiometry was also validated on SDS-PAGE
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Elongator complex protein 1 / ELP1 / IkappaB kinase complex-associated protein / IKK complex-associated protein


Mass: 149756.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Elp1, Ikap, Ikbkap / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7TT37
#2: Protein Elongator complex protein 2 / ELP2 / STAT3-interacting protein 1 / StIP1


Mass: 93193.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Elp2, Statip1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q91WG4
#3: Protein Elongator complex protein 3 / tRNA uridine(34) acetyltransferase


Mass: 62481.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Elp3 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9CZX0, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
#4: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Mouse Elp123 with bound SAM / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.6100 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
220 mMHEPESHEPES1
35 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 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 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: 6415
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.2.0particle selectionblob, TOPAZ
2EPU2.1image acquisition
4cryoSPARCv3.2.0CTF correctionpatch CTF
7UCSF ChimeraXmodel fitting
9cryoSPARCv3.2.0initial Euler assignmentAb-Initio Reconstruction
10RELION3.1final Euler assignment3D auto-refine
11RELION3.1classificationmasked 3D classification
12RELION3.13D reconstructionPostprocessing
13PHENIXmodel refinement
Image processingDetails: 20 eV slit, fully tuned before the experiment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 185923
Details: given number of particles is after TOPAZ picking, 2D cleaning and duplicate removal
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42894
Details: Declared resolution for the post-process filtered map
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: After initial rigid body fit, further fitting was done using NAMDINATOR with manual corrections in COOT
Atomic model buildingPDB-ID: 6QK7

6qk7


Accession code: 6QK7 / Source name: PDB / Type: experimental model

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