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- EMDB-15635: Cryo-EM structure of yeast Elp456 subcomplex -

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Basic information

Entry
Database: EMDB / ID: EMD-15635
TitleCryo-EM structure of yeast Elp456 subcomplex
Map data
Sample
  • Complex: Yeast Elp456 subcomplex
    • Protein or peptide: Elongator complex protein 4
    • Protein or peptide: Elongator complex protein 5
    • Protein or peptide: Elongator complex protein 6
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)
Methodsingle 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, 2 items
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, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15635.png

Downloads & links

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Map

FileDownload / File: emd_15635.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0242
Minimum - Maximum-0.035762765 - 0.07653746
Average (Standard dev.)9.877364e-05 (±0.0017283901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15635_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15635_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast Elp456 subcomplex

EntireName: Yeast Elp456 subcomplex
Components
  • Complex: Yeast Elp456 subcomplex
    • Protein or peptide: Elongator complex protein 4
    • Protein or peptide: Elongator complex protein 5
    • Protein or peptide: Elongator complex protein 6

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Supramolecule #1: Yeast Elp456 subcomplex

SupramoleculeName: Yeast Elp456 subcomplex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 233.8 KDa

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Macromolecule #1: Elongator complex protein 4

MacromoleculeName: Elongator complex protein 4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 51.232469 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSFRKRGEIL NDRGSGLRGP LLRGPPRTSS TPLRTGNRRA PGNVPLSDTT ARLKKLNIAD ESKTKMGLDS SHVGVRPSPA TSQPTTSTG SADLDSILGH MGLPLGNSVL VEEQSTTEFH SILGKLFAAQ GIVHNRISDS SADKTRNGDT HVIVLSLNQM F AKELPGIY ...String:
MSFRKRGEIL NDRGSGLRGP LLRGPPRTSS TPLRTGNRRA PGNVPLSDTT ARLKKLNIAD ESKTKMGLDS SHVGVRPSPA TSQPTTSTG SADLDSILGH MGLPLGNSVL VEEQSTTEFH SILGKLFAAQ GIVHNRISDS SADKTRNGDT HVIVLSLNQM F AKELPGIY KGSRKQMKKN LISEEESKVT VQNLNETQRS TPSRYKDLKI AWKYKLADEK RLGSPDRDDI QQNSEYKDYN HQ FDITTRL MPAPIASELT FIAPTQPVST ILSQIEQTIK RNDKKLIRIV IPSLLHPAMY PPKMFESSEI IGLMHGVRSL VKK YYERVV LFASISIDII TPPLLVLLRN MFDSVINLEP FNQEMTEFLE RVYKSQPGKI QHGLVHILKL PVFTDRGEMR VLKS EWAFK NGRKKFEIEQ WGIPVDDAEG SAASEQSHSH SHSDEISHNI PAKKTKISLD Y

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Macromolecule #2: Elongator complex protein 5

MacromoleculeName: Elongator complex protein 5 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 35.252496 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MASSSHNPVI LLKRILSLTE SSPFILCLDS IAQTSYKLIQ EFVHQSKSKG NEYPIVYISF ETVNKPSYCT QFIDATQMDF VHLVKQIIS YLPAATATQA KKHMVIIDSL NYISTEYITR FLSEIASPHC TMVATYHKDI KDENRTVIPD WNNNYPDKLT L LQFMATTI ...String:
MASSSHNPVI LLKRILSLTE SSPFILCLDS IAQTSYKLIQ EFVHQSKSKG NEYPIVYISF ETVNKPSYCT QFIDATQMDF VHLVKQIIS YLPAATATQA KKHMVIIDSL NYISTEYITR FLSEIASPHC TMVATYHKDI KDENRTVIPD WNNNYPDKLT L LQFMATTI VDIDVVLTGT LDTEEVSELL NEFRIPRGLN NDIFQLRLVN KRKSGRSLEY DFIVNSNTHE YELLSTTKQE EE SSSNGLE TPEMLQGLTT FNLGTSNKQK LAKDQVALPF LEAQSFGQGG AIVYEYEKDD DYDEEDPYED PF

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Macromolecule #3: Elongator complex protein 6

MacromoleculeName: Elongator complex protein 6 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 30.602611 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGSVQRQDLV LFSDQSVLPA HFFQDSNSHN LFFITHQSCT QPLWMINALV ETHVLGSPSS LNESSSSMLP SSTRSHAVLA SFIHEQNYF TNSLNKLKIP SNNYNVLDFL SDFIVNNIHN KPRDKILSDV LAKFSAAIQN NPTDTIVIIE QPELLLSLVS G LTCSELNN ...String:
MGSVQRQDLV LFSDQSVLPA HFFQDSNSHN LFFITHQSCT QPLWMINALV ETHVLGSPSS LNESSSSMLP SSTRSHAVLA SFIHEQNYF TNSLNKLKIP SNNYNVLDFL SDFIVNNIHN KPRDKILSDV LAKFSAAIQN NPTDTIVIIE QPELLLSLVS G LTCSELNN KFITPLLRQC KVLIIVSNSD IFNIDEYDAS VHSSNLQNFY KSSFIKSMIN LNLNPLKTGF AKDVTGSLHV CR GGAPIAT SNTSLHVVEN EYLYLNEKES TKLFYR

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPESHEPES
3.0 mMDithiothreitolDithiothreitol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 15 s wait time, blot force 5, 5 s blot time.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 4716 / Average exposure time: 1.82 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 364424
Details: given number of particles is after TOPAZ picking and 2D cleaning
Startup modelType of model: INSILICO MODEL
In silico model: SWISS-MODEL generated full length Elp456 based on 4A8J structure
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2.0) / Software - details: Ab-Initio Reconstruction / Details: Ab-Initio Reconstruction
Final 3D classificationNumber classes: 5 / Avg.num./class: 12500 / Software - Name: RELION (ver. 3.1) / Software - details: masked 3D classification
Details: masked 3D classification with two classes of nice Elp456 density, each approx. 65k ptcls; and 3 remaining classes, each approx. 33k ptcls
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Software - details: 3D auto-refine / Details: 3D auto-refine
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Software - details: Postprocessing
Details: Given global resolution estimation for post-process sharpened map
Number images used: 128593
Details20 eV slit, fully tuned before the experiment
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4a8j

DetailsAfter initial rigid body fit, further fitting was done using NAMDINATOR
RefinementProtocol: RIGID BODY FIT
Output model

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

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