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- EMDB-4574: tRNA-bound Elongator catalytic subcomplex, tRNA-Elp123 lobe. -

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Basic information

Entry
Database: EMDB / ID: EMD-4574
TitletRNA-bound Elongator catalytic subcomplex, tRNA-Elp123 lobe.
Map dataPostprocessed map of tRNA-bound Elongator catalytic subcomplex, Elp123lobe-tRNA from yeast at 4.4 A resolution.
Sample
  • Complex: tRNA-bound Elongator catalytic subcomplex lobe, tRNA-Elp123 lobe
    • Protein or peptide: Elongator complex protein 1, Elp1
    • Protein or peptide: Elongator complex protein 2, Elp2
    • Protein or peptide: Elongator complex protein 3, Elp3
    • RNA: tRNA Ala UGC
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsDauden MI / Weis F
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research FoundationBR921/9-1 & Mu3173/2-1 Germany
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 30, 2019-
Header (metadata) releaseFeb 6, 2019-
Map releaseJul 17, 2019-
UpdateJul 31, 2019-
Current statusJul 31, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0459
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0459
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4574.png

Downloads & links

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Map

FileDownload / File: emd_4574.map.gz / Format: CCP4 / Size: 190.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map of tRNA-bound Elongator catalytic subcomplex, Elp123lobe-tRNA from yeast at 4.4 A resolution.
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.0459 / Movie #1: 0.0459
Minimum - Maximum-0.06273258 - 0.21938784
Average (Standard dev.)0.00020899644 (±0.0030904189)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions368368368
Spacing368368368
CellA=B=C: 496.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z496.800496.800496.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS368368368
D min/max/mean-0.0630.2190.000

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Supplemental data

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Half map: Half map of tRNA-bound Elongator catalytic subcomplex, Elp123lobe-tRNA...

Fileemd_4574_half_map_1.map
AnnotationHalf map of tRNA-bound Elongator catalytic subcomplex, Elp123lobe-tRNA from yeast.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of tRNA-bound Elongator catalytic subcomplex, Elp123lobe-tRNA...

Fileemd_4574_half_map_2.map
AnnotationHalf map of tRNA-bound Elongator catalytic subcomplex, Elp123lobe-tRNA from yeast.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : tRNA-bound Elongator catalytic subcomplex lobe, tRNA-Elp123 lobe

EntireName: tRNA-bound Elongator catalytic subcomplex lobe, tRNA-Elp123 lobe
Components
  • Complex: tRNA-bound Elongator catalytic subcomplex lobe, tRNA-Elp123 lobe
    • Protein or peptide: Elongator complex protein 1, Elp1
    • Protein or peptide: Elongator complex protein 2, Elp2
    • Protein or peptide: Elongator complex protein 3, Elp3
    • RNA: tRNA Ala UGC

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Supramolecule #1: tRNA-bound Elongator catalytic subcomplex lobe, tRNA-Elp123 lobe

SupramoleculeName: tRNA-bound Elongator catalytic subcomplex lobe, tRNA-Elp123 lobe
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The EM map corresponds to one lobe of the Elp123 complex bound to one tRNA Ala UGC.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 643 KDa

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Macromolecule #1: Elongator complex protein 1, Elp1

MacromoleculeName: Elongator complex protein 1, Elp1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVEHDKSGSK RQELRSNMRN LITLNKGKFK PTASTAEGDE DDLSFTLLDS VFDTLSDSIT CVLGSTDIG AIEVQQFMKD GSRNVLASFN IQTFDDKLLS FVHFADINQL VFVFEQGDII T ATYDPVSL DPAETLIEIM GTIDNGIAAA QWSYDEETLA MVTKDRNVVV ...String:
MVEHDKSGSK RQELRSNMRN LITLNKGKFK PTASTAEGDE DDLSFTLLDS VFDTLSDSIT CVLGSTDIG AIEVQQFMKD GSRNVLASFN IQTFDDKLLS FVHFADINQL VFVFEQGDII T ATYDPVSL DPAETLIEIM GTIDNGIAAA QWSYDEETLA MVTKDRNVVV LSKLFEPISE YH LEVDDLK ISKHVTVGWG KKETQFRGKG ARAMEREALA SLKASGLVGN QLRDPTMPYM VDT GDVTAL DSHEITISWR GDCDYFAVSS VEEVPDEDDE TKSIKRRAFR VFSREGQLDS ASEP VTGME HQLSWKPQGS LIASIQRKTD LGEEDSVDVI FFERNGLRHG EFDTRLPLDE KVESV CWNS NSEALAVVLA NRIQLWTSKN YHWYLKQELY ASDISYVKWH PEKDFTLMFS DAGFIN IVD FAYKMAQGPT LEPFDNGTSL VVDGRTVNIT PLALANVPPP MYYRDFETPG NVLDVAC SF SNEIYAAINK DVLIFAAVPS IEEMKKGKHP SIVCEFPKSE FTSEVDSLRQ VAFINDSI V GVLLDTDNLS RIALLDIQDI TQPTLITIVE VYDKIVLLRS DFDYNHLVYE TRDGTVCQL DAEGQLMEIT KFPQLVRDFR VKRVHNTSAE DDDNWSAESS ELVAFGITNN GKLFANQVLL ASAVTSLEI TDSFLLFTTA QHNLQFVHLN STDFKPLPLV EEGVEDERVR AIERGSILVS V IPSKSSVV LQATRGNLET IYPRIMVLAE VRKNIMAKRY KEAFIVCRTH RINLDILHDY AP ELFIENL EVFINQIGRV DYLNLFISCL SEDDVTKTKY KETLYSGISK SFGMEPAPLT EMQ IYMKKK MFDPKTSKVN KICDAVLNVL LSNPEYKKKY LQTIITAYAS QNPQNLSAAL KLIS ELENS EEKDSCVTYL CFLQDVNVVY KSALSLYDVS LALLVAQKSQ MDPREYLPFL QELQD NEPL RRKFLIDDYL GNYEKALEHL SEIDKDGNVS EEVIDYVESH DLYKHGLALY RYDSEK QNV IYNIYAKHLS SNQMYTDAAV AYEMLGKLKE AMGAYQSAKR WREAMSIAVQ KFPEEVE SV AEELISSLTF EHRYVDAADI QLEYLDNVKE AVALYCKAYR YDIASLVAIK AKKDELLE E VVDPGLGEGF GIIAELLADC KGQINSQLRR LRELRAKKEE NPYAFYGQET EQADDVSVA PSETSTQESF FTRYTGKTGG TAKTGASRRT AKNKRREERK RARGKKGTIY EEEYLVQSVG RLIERLNQT KPDAVRVVEG LCRRNMREQA HQIQKNFVEV LDLLKANVKE IYSISEKDRE R VNENGEVY YIPEIPVPEI HDFPKSHIVD F

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Macromolecule #2: Elongator complex protein 2, Elp2

MacromoleculeName: Elongator complex protein 2, Elp2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVECITPEAI FIGANKQTQV SDIHKVKKIV AFGAGKTIAL WDPIEPNNKG VYATLKGHEA EVTCVRFVP DSDFMVSASE DHHVKIWKFT DYSHLQCIQT IQHYSKTIVA LSALPSLISV G CADGTISI WRQNIQNDEF GLAHEFTIKK GFFYPLCLSL SKVEEKKYLL ...String:
MVECITPEAI FIGANKQTQV SDIHKVKKIV AFGAGKTIAL WDPIEPNNKG VYATLKGHEA EVTCVRFVP DSDFMVSASE DHHVKIWKFT DYSHLQCIQT IQHYSKTIVA LSALPSLISV G CADGTISI WRQNIQNDEF GLAHEFTIKK GFFYPLCLSL SKVEEKKYLL AIGGTNVNVF IA SFILSDS GIEKCRVVAE LEGHEDWVKS LAFRHQETPG DYLLCSGSQD RYIRLWRIRI NDL IDDSEE DSKKLTLLSN KQYKFQIDDE LRVGINFEAL IMGHDDWISS LQWHESRLQL LAAT ADTSL MVWEPDETSG IWVCSLRLGE MSSKGASTAT GSSGGFWSCL WFTHERMDFF LTNGK TGSW RMWATKDNII CDQRLGISGA TKDVTDIAWS PSGEYLLATS LDQTTRLFAP WIYDAS GRK REIATWHEFS RPQIHGYDMI CVETVTDTRF VSGGDEKILR SFDLPKGVAG MLQKFVG IQ FEEKSEMPDS ATVPVLGLSN KAGEDDANED DEEEEGGNKE TPDITDPLSL LECPPMED Q LQRHLLWPEV EKLYGHGFEI TCLDISPDQK LIASACRSNN VQNAVIRIFS TENWLEIKP ALPFHSLTIT RLKFSKDGKF LLSVCRDRKW ALWERNMEDN TFELRFKNEK PHTRIIWDAD WAPLEFGNV FVTASRDKTV KVWRHQKEPA DDYVLEASIK HTKAVTAISI HDSMIREKIL I SVGLENGE IYLYSYTLGK FELITQLNED ITPADKITRL RWSHLKRNGK LFLGVGSSDL ST RIYSLAY E

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Macromolecule #3: Elongator complex protein 3, Elp3

MacromoleculeName: Elongator complex protein 3, Elp3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MARHGKGPKT NKKKLAPEKE RFIQCCADIT LELTDSLTSG TTREINLNGL ITKYSKKYKL KQQPRLTDI INSIPDQYKK YLLPKLKAKP VRTASGIAVV AVMCKPHRCP HIAYTGNICV Y CPGGPDSD FEYSTQSYTG YEPTSMRAIR ARYDPYEQAR GRVEQLKQLG ...String:
MARHGKGPKT NKKKLAPEKE RFIQCCADIT LELTDSLTSG TTREINLNGL ITKYSKKYKL KQQPRLTDI INSIPDQYKK YLLPKLKAKP VRTASGIAVV AVMCKPHRCP HIAYTGNICV Y CPGGPDSD FEYSTQSYTG YEPTSMRAIR ARYDPYEQAR GRVEQLKQLG HSIDKVEYVL MG GTFMSLP KEYREDFIVK LHNALSGFNG NDIDEAILYS QQSLTKCVGI TIETRPDYCT QTH LDDMLK YGCTRLEIGV QSLYEDVARD TNRGHTVRSV CETFAVSKDA GYKVVSHMMP DLPN VGMER DIEQFKEYFE NPDFRTDGLK IYPTLVIRGT GLYELWKTGR YKSYSANALV DLVAR ILAL VPPWTRIYRV QRDIPMPLVT SGVDNGNLRE LALARMKDLG TTCRDVRTRE VGIQEV HHK VQPDQVELIR RDYYANGGWE TFLSYEDPKK DILIGLLRLR KASKKYTYRK EFTSQRT SI VRELHVYGSV VPLHSRDPRK FQHQGFGTLL MEEAERIAKE EHGSEKISVI SGVGVRNY Y GKLGYELDGP YMSKRI

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Macromolecule #4: tRNA Ala UGC

MacromoleculeName: tRNA Ala UGC / type: rna / ID: 4
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
GGGCACAUGG CGCAGUUGGU AGCGCGCUUC CCUUGCAAGG AAGAGGUCAU CGGUUCGAUU CCGGUUGCGU CCA

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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
10.0 mMHepesHepes
125.0 mMNaClSodium chlorideSodium chloride
50.0 mMNaFSodium fluoride
0.1 mMNa3VO4Sodium vanadate
5.0 mMC4H10O2S2Dithiothreitol
5.0 mMMgCl2Magnesium chloride

Details: 1 mM acetyl-CoA was added when incubating the Elp123 and the tRNA.
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Details: Pelco EasyGlow glow discharger
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 2.5 ul of sample, blotting parameters: wait time 15 s, blot force 5, blot time 5-8 s..
DetailsPure Elp123 was incubated with in vitro transcribed tRNA Ala UGC at 1:5 molar ratio for 30 min at 25 degrees in the presence of 1 mM acetyl-CoA, and then immediately plunged.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
DetailsGatan Quantum energy filter and a K2 Summit direct detector
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 6990 / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1500000
Details: Initially 10888 particles were manually selected using EMAN2 boxer swarm tool, and used as 2D templates for the autopicking procedure in relion, that yielded 1.5 million particles.
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

4151


Details: The initial model was low pass filtered to 60 A.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 32500 / Software - Name: RELION (ver. 2)
Details: Focused 3D classification was performed after partial signal subtraction of the lobe.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 23020
DetailsThe detector was operated in super resolution mode.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5m2n

chain_id: B

5cqs

chain_id: A

5cqs

chain_id: D, residue_range: 932-1349
RefinementProtocol: FLEXIBLE FIT

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