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- PDB-6qk7: Elongator catalytic subcomplex Elp123 lobe -

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Basic information

Entry
Database: PDB / ID: 6qk7
TitleElongator catalytic subcomplex Elp123 lobe
Components
  • Elongator complex protein 1
  • Elongator complex protein 2
  • Elongator complex protein 3
KeywordsTRANSLATION / Elongator / yeast / tRNA modification / Elp123
Function / homology
Function and homology information


tRNA uridine(34) acetyltransferase activity / elongator holoenzyme complex / tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation / protein urmylation / tRNA wobble uridine modification / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / : / protein transport / regulation of translation / 4 iron, 4 sulfur cluster binding ...tRNA uridine(34) acetyltransferase activity / elongator holoenzyme complex / tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation / protein urmylation / tRNA wobble uridine modification / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / : / 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 / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Elongator complex protein 1 / Elongator complex protein 2 / IKI3 family / 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 ...Elongator complex protein 1 / Elongator complex protein 2 / IKI3 family / 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 / Acetyltransferase (GNAT) family / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain / Acyl-CoA N-acyltransferase / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
5'-DEOXYADENOSINE / IRON/SULFUR CLUSTER / Elongator complex protein 2 / Elongator complex protein 3 / Elongator complex protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDauden, M.I. / Jaciuk, M. / Glatt, S.
Funding support Germany, Poland, 2items
OrganizationGrant numberCountry
German Research FoundationBR921/9-1 & Mu3173/2-1 Germany
Polish National Science CentreUMO-2015/19/B/NZ1/00343 Poland
CitationJournal: Sci Adv / Year: 2019
Title: Molecular basis of tRNA recognition by the Elongator complex.
Authors: Maria I Dauden / Marcin Jaciuk / Felix Weis / Ting-Yu Lin / Carolin Kleindienst / Nour El Hana Abbassi / Heena Khatter / Rościsław Krutyhołowa / Karin D Breunig / Jan Kosinski / Christoph ...Authors: Maria I Dauden / Marcin Jaciuk / Felix Weis / Ting-Yu Lin / Carolin Kleindienst / Nour El Hana Abbassi / Heena Khatter / Rościsław Krutyhołowa / Karin D Breunig / Jan Kosinski / Christoph W Müller / Sebastian Glatt /
Abstract: The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of ...The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo-electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer.
History
DepositionJan 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 17, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4571
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)460,2106
Polymers459,6074
Non-polymers6032
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12280 Å2
ΔGint-49 kcal/mol
Surface area116960 Å2
MethodPISA

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Components

#1: Protein Elongator complex protein 1 / Gamma-toxin target 1 / Protein IKI3


Mass: 153166.266 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: Chain D corresponds to the C-terminal domain of Elp1
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q06706
#2: Protein Elongator complex protein 2 / Gamma-toxin target 2


Mass: 89519.430 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P42935
#3: Protein Elongator complex protein 3 / Gamma-toxin target 3


Mass: 63755.059 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: FeS cluster and 5DA
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q02908, histone acetyltransferase
#4: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#5: Chemical ChemComp-5AD / 5'-DEOXYADENOSINE / Deoxyadenosine


Mass: 251.242 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H13N5O3

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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: Elongator catalytic subcomplex Elp123 / Type: COMPLEX
Details: The EM map corresponds to one lobe of the Elp123 complex, that includes one copy of Elp1, Elp2 and Elp3, and the C-terminal part of a second copy of Elp1.
Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.621 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepesHepes1
2125 mMSodium chlorideNaClSodium chloride1
350 mMSodium fluorideNaF1
40.1 mMSodium vanadateNa3VO41
55 mMbeta-mercaptoethanol2-MercaptoethanolC2H6OS1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was cross-linked with 0.01% glutaraldehyde, quenched and then plunged.
Specimen supportDetails: Pelco EasyGlow glow discharger, 20 mA / Grid 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: 2.5 ul of sample, blotting parameters: wait time 15 s, blot force 5, blot time 5-8 s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Gatan Quantum energy filter and a K2 Summit direct detector
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 43 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4614
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
2RELIONparticle selectionautopicking procedure
3SerialEMimage acquisition
5CTFFIND4CTF correction
8UCSF Chimeramodel fittingFit in map tool
9iMODFIT1.44model fitting
11RSRefmodel refinement
12RELION2initial Euler assignment
13RELION2final Euler assignment
15RELION23D reconstruction
Image processingDetails: The detector was operated in super resolution mode.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1000000
Details: Initially 8563 particles were manually selected using EMAN2 boxer swarm tool, and used as 2D templates for the autopicking procedure in relion, that yielded 1 million particles.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84135 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1FLEXIBLE FITREAL
2
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
15M2N

5m2n

B1
25CQS

5cqs

A1932-1349
35CQS

5cqs

D2

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