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- EMDB-15623: Cryo-EM structure of yeast Elp123 in complex with alanine tRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-15623
TitleCryo-EM structure of yeast Elp123 in complex with alanine tRNA
Map data
Sample
  • Complex: Yeast Elp123 in complex with alanine tRNA
    • Protein or peptide: Elongator complex protein 1
    • Protein or peptide: Elongator complex protein 2
    • Protein or peptide: Elongator complex protein 3
    • RNA: Alanine tRNA
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: 5'-DEOXYADENOSINE
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 / : / protein transport / regulation of translation / 4 iron, 4 sulfur cluster binding ...tRNA carboxymethyluridine synthase / tRNA uridine(34) acetyltransferase activity / elongator holoenzyme complex / tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation / protein urmylation / tRNA wobble uridine modification / : / 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 / 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 / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Elongator complex protein 2 / Elongator complex protein 3 / Elongator complex protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.96 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 21, 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_15623.png

Downloads & links

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Map

FileDownload / File: emd_15623.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.81 Å
Density
Contour LevelBy AUTHOR: 0.00855
Minimum - Maximum-0.02068874 - 0.06225855
Average (Standard dev.)0.00010923615 (±0.0010637707)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 340.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15623_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_15623_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 Elp123 in complex with alanine tRNA

EntireName: Yeast Elp123 in complex with alanine tRNA
Components
  • Complex: Yeast Elp123 in complex with alanine tRNA
    • Protein or peptide: Elongator complex protein 1
    • Protein or peptide: Elongator complex protein 2
    • Protein or peptide: Elongator complex protein 3
    • RNA: Alanine tRNA
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: 5'-DEOXYADENOSINE

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Supramolecule #1: Yeast Elp123 in complex with alanine tRNA

SupramoleculeName: Yeast Elp123 in complex with alanine tRNA / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 636.1 KDa

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Macromolecule #1: Elongator complex protein 1

MacromoleculeName: Elongator complex protein 1 / 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: 153.166266 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVEHDKSGSK RQELRSNMRN LITLNKGKFK PTASTAEGDE DDLSFTLLDS VFDTLSDSIT CVLGSTDIGA IEVQQFMKDG SRNVLASFN IQTFDDKLLS FVHFADINQL VFVFEQGDII TATYDPVSLD PAETLIEIMG TIDNGIAAAQ WSYDEETLAM V TKDRNVVV ...String:
MVEHDKSGSK RQELRSNMRN LITLNKGKFK PTASTAEGDE DDLSFTLLDS VFDTLSDSIT CVLGSTDIGA IEVQQFMKDG SRNVLASFN IQTFDDKLLS FVHFADINQL VFVFEQGDII TATYDPVSLD PAETLIEIMG TIDNGIAAAQ WSYDEETLAM V TKDRNVVV LSKLFEPISE YHLEVDDLKI SKHVTVGWGK KETQFRGKGA RAMEREALAS LKASGLVGNQ LRDPTMPYMV DT GDVTALD SHEITISWRG DCDYFAVSSV EEVPDEDDET KSIKRRAFRV FSREGQLDSA SEPVTGMEHQ LSWKPQGSLI ASI QRKTDL GEEDSVDVIF FERNGLRHGE FDTRLPLDEK VESVCWNSNS EALAVVLANR IQLWTSKNYH WYLKQELYAS DISY VKWHP EKDFTLMFSD AGFINIVDFA YKMAQGPTLE PFDNGTSLVV DGRTVNITPL ALANVPPPMY YRDFETPGNV LDVAC SFSN EIYAAINKDV LIFAAVPSIE EMKKGKHPSI VCEFPKSEFT SEVDSLRQVA FINDSIVGVL LDTDNLSRIA LLDIQD ITQ PTLITIVEVY DKIVLLRSDF DYNHLVYETR DGTVCQLDAE GQLMEITKFP QLVRDFRVKR VHNTSAEDDD NWSAESS EL VAFGITNNGK LFANQVLLAS AVTSLEITDS FLLFTTAQHN LQFVHLNSTD FKPLPLVEEG VEDERVRAIE RGSILVSV I PSKSSVVLQA TRGNLETIYP RIMVLAEVRK NIMAKRYKEA FIVCRTHRIN LDILHDYAPE LFIENLEVFI NQIGRVDYL NLFISCLSED DVTKTKYKET LYSGISKSFG MEPAPLTEMQ IYMKKKMFDP KTSKVNKICD AVLNVLLSNP EYKKKYLQTI ITAYASQNP QNLSAALKLI SELENSEEKD SCVTYLCFLQ DVNVVYKSAL SLYDVSLALL VAQKSQMDPR EYLPFLQELQ D NEPLRRKF LIDDYLGNYE KALEHLSEID KDGNVSEEVI DYVESHDLYK HGLALYRYDS EKQNVIYNIY AKHLSSNQMY TD AAVAYEM LGKLKEAMGA YQSAKRWREA MSIAVQKFPE EVESVAEELI SSLTFEHRYV DAADIQLEYL DNVKEAVALY CKA YRYDIA SLVAIKAKKD ELLEEVVDPG LGEGFGIIAE LLADCKGQIN SQLRRLRELR AKKEENPYAF YGQETEQADD VSVA PSETS TQESFFTRYT GKTGGTAKTG ASRRTAKNKR REERKRARGK KGTIYEEEYL VQSVGRLIER LNQTKPDAVR VVEGL CRRN MREQAHQIQK NFVEVLDLLK ANVKEIYSIS EKDRERVNEN GEVYYIPEIP VPEIHDFPKS HIVDF

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Macromolecule #2: Elongator complex protein 2

MacromoleculeName: Elongator complex protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 89.51943 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVECITPEAI FIGANKQTQV SDIHKVKKIV AFGAGKTIAL WDPIEPNNKG VYATLKGHEA EVTCVRFVPD SDFMVSASED HHVKIWKFT DYSHLQCIQT IQHYSKTIVA LSALPSLISV GCADGTISIW RQNIQNDEFG LAHEFTIKKG FFYPLCLSLS K VEEKKYLL ...String:
MVECITPEAI FIGANKQTQV SDIHKVKKIV AFGAGKTIAL WDPIEPNNKG VYATLKGHEA EVTCVRFVPD SDFMVSASED HHVKIWKFT DYSHLQCIQT IQHYSKTIVA LSALPSLISV GCADGTISIW RQNIQNDEFG LAHEFTIKKG FFYPLCLSLS K VEEKKYLL AIGGTNVNVF IASFILSDSG IEKCRVVAEL EGHEDWVKSL AFRHQETPGD YLLCSGSQDR YIRLWRIRIN DL IDDSEED SKKLTLLSNK QYKFQIDDEL RVGINFEALI MGHDDWISSL QWHESRLQLL AATADTSLMV WEPDETSGIW VCS LRLGEM SSKGASTATG SSGGFWSCLW FTHERMDFFL TNGKTGSWRM WATKDNIICD QRLGISGATK DVTDIAWSPS GEYL LATSL DQTTRLFAPW IYDASGRKRE IATWHEFSRP QIHGYDMICV ETVTDTRFVS GGDEKILRSF DLPKGVAGML QKFVG IQFE EKSEMPDSAT VPVLGLSNKA GEDDANEDDE EEEGGNKETP DITDPLSLLE CPPMEDQLQR HLLWPEVEKL YGHGFE ITC LDISPDQKLI ASACRSNNVQ NAVIRIFSTE NWLEIKPALP FHSLTITRLK FSKDGKFLLS VCRDRKWALW ERNMEDN TF ELRFKNEKPH TRIIWDADWA PLEFGNVFVT ASRDKTVKVW RHQKEPADDY VLEASIKHTK AVTAISIHDS MIREKILI S VGLENGEIYL YSYTLGKFEL ITQLNEDITP ADKITRLRWS HLKRNGKLFL GVGSSDLSTR IYSLAYE

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Macromolecule #3: Elongator complex protein 3

MacromoleculeName: Elongator complex protein 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 63.755059 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MARHGKGPKT NKKKLAPEKE RFIQCCADIT LELTDSLTSG TTREINLNGL ITKYSKKYKL KQQPRLTDII NSIPDQYKKY LLPKLKAKP VRTASGIAVV AVMCKPHRCP HIAYTGNICV YCPGGPDSDF EYSTQSYTGY EPTSMRAIRA RYDPYEQARG R VEQLKQLG ...String:
MARHGKGPKT NKKKLAPEKE RFIQCCADIT LELTDSLTSG TTREINLNGL ITKYSKKYKL KQQPRLTDII NSIPDQYKKY LLPKLKAKP VRTASGIAVV AVMCKPHRCP HIAYTGNICV YCPGGPDSDF EYSTQSYTGY EPTSMRAIRA RYDPYEQARG R VEQLKQLG HSIDKVEYVL MGGTFMSLPK EYREDFIVKL HNALSGFNGN DIDEAILYSQ QSLTKCVGIT IETRPDYCTQ TH LDDMLKY GCTRLEIGVQ SLYEDVARDT NRGHTVRSVC ETFAVSKDAG YKVVSHMMPD LPNVGMERDI EQFKEYFENP DFR TDGLKI YPTLVIRGTG LYELWKTGRY KSYSANALVD LVARILALVP PWTRIYRVQR DIPMPLVTSG VDNGNLRELA LARM KDLGT TCRDVRTREV GIQEVHHKVQ PDQVELIRRD YYANGGWETF LSYEDPKKDI LIGLLRLRKA SKKYTYRKEF TSQRT SIVR ELHVYGSVVP LHSRDPRKFQ HQGFGTLLME EAERIAKEEH GSEKISVISG VGVRNYYGKL GYELDGPYMS KRI

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Macromolecule #4: Alanine tRNA

MacromoleculeName: Alanine tRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 23.538932 KDa
SequenceString:
GGGCACAUGG CGCAGUUGGU AGCGCGCUUC CCUUGCAAGG AAGAGGUCAU CGGUUCGAUU CCGGUUGCGU CCA

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #6: 5'-DEOXYADENOSINE

MacromoleculeName: 5'-DEOXYADENOSINE / type: ligand / ID: 6 / Number of copies: 1 / Formula: 5AD
Molecular weightTheoretical: 251.242 Da
Chemical component information

ChemComp-5AD:
5'-DEOXYADENOSINE

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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 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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number real images: 11080 / Average electron dose: 42.45 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 172149
Details: given number of particles is after TOPAZ picking and Ab-Initio classification
Startup modelType of model: INSILICO MODEL
In silico model: SWISS-MODEL generated full length Elp123 and alanine tRNA built from PDB ID 1EHZ
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Software - details: Postprocessing / Number images used: 16809
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2.0) / Software - details: Ab-Initio Reconstruction / Details: Ab-Initio Reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Software - details: 3D auto-refine / Details: 3D auto-refine
Final 3D classificationNumber classes: 3 / Avg.num./class: 48660 / Software - Name: RELION (ver. 3.1) / Software - details: masked 3D classification / Details: masked 3D classification to improve tRNA density
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6qk7

,

1ehz

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

PDB-8asw:
Cryo-EM structure of yeast Elp123 in complex with alanine tRNA

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