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- EMDB-6615: State 1 of cryo-EM structure of the yeast pre-60S particles isola... -

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Basic information

Entry
Database: EMDB / ID: EMD-6615
TitleState 1 of cryo-EM structure of the yeast pre-60S particles isolated with Nog2-TAP
Map dataReconstruction of pre-60S ribosomal subunit
Sample
  • Sample: state1 of pre-60S ribosome subunit
  • Complex: pre-60S ribosome
KeywordsPre-60S particle / ribosome biogenesis / assembly factor / Nog2 / Nog1 / ITS2 / Translation
Function / homology
Function and homology information


protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear exosome (RNase complex) / Hydrolases / PeBoW complex / Antigen processing: Ubiquitination & Proteasome degradation / 7S RNA binding / rRNA primary transcript binding / positive regulation of ATP-dependent activity ...protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear exosome (RNase complex) / Hydrolases / PeBoW complex / Antigen processing: Ubiquitination & Proteasome degradation / 7S RNA binding / rRNA primary transcript binding / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear-transcribed mRNA catabolic process / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of 5.8S rRNA / proteasome binding / ribosomal subunit export from nucleus / Major pathway of rRNA processing in the nucleolus and cytosol / SRP-dependent cotranslational protein targeting to membrane / 90S preribosome / GTP hydrolysis and joining of the 60S ribosomal subunit / ATPase activator activity / Formation of a pool of free 40S subunits / ribosomal large subunit binding / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / negative regulation of mRNA splicing, via spliceosome / protein-RNA complex assembly / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal large subunit export from nucleus / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / ribonucleoprotein complex binding / maturation of SSU-rRNA / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / Neutrophil degranulation / translation initiation factor activity / nuclear periphery / small-subunit processome / proteasome complex / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / maintenance of translational fidelity / macroautophagy / protein catabolic process / ribosomal large subunit assembly / rRNA processing / metallopeptidase activity / large ribosomal subunit rRNA binding / protein-macromolecule adaptor activity / protein transport / ribosome biogenesis / cytoplasmic translation / 5S rRNA binding / cytosolic large ribosomal subunit / ATPase binding / negative regulation of translation / nucleic acid binding / rRNA binding / ribosome / structural constituent of ribosome / translation / mRNA binding / GTPase activity / GTP binding / nucleolus / proteolysis / RNA binding / zinc ion binding / nucleoplasm / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ribosome biogenesis protein Rpf2 / Ribosome biogenesis protein 15, RNA recognition motif / : / Ribosome biogenesis protein Nop53/GLTSCR2 / Nop53 (60S ribosomal biogenesis) / Cgr1-like / Cgr1 family / Ribosomal biogenesis regulatory protein / Ribosome biogenesis regulatory protein (RRS1) / Nucleolar GTP-binding protein 2, N-terminal domain ...Ribosome biogenesis protein Rpf2 / Ribosome biogenesis protein 15, RNA recognition motif / : / Ribosome biogenesis protein Nop53/GLTSCR2 / Nop53 (60S ribosomal biogenesis) / Cgr1-like / Cgr1 family / Ribosomal biogenesis regulatory protein / Ribosome biogenesis regulatory protein (RRS1) / Nucleolar GTP-binding protein 2, N-terminal domain / Domain of unknown function DUF2423 / Nucleolar GTP-binding protein 2 / NGP1NT (NUC091) domain / Protein of unknown function (DUF2423) / NLE / NLE (NUC135) domain / Guanine nucleotide-binding protein-like 3, N-terminal domain / GNL3L/Grn1 putative GTPase / Zinc-finger double-stranded RNA-binding / Zinc finger, double-stranded RNA binding / Pescadillo / Pescadillo N-terminus / GTP-binding protein, orthogonal bundle domain superfamily / Ribosomal biogenesis NSA2 family / BRCT domain, a BRCA1 C-terminus domain / Ribosome assembly factor Mrt4 / NOG, C-terminal / Nucleolar GTP-binding protein 1 / NOGCT (NUC087) domain / Nucleolar GTP-binding protein 1, Rossman-fold domain / NOG1, N-terminal helical domain / Nucleolar GTP-binding protein 1 (NOG1) / NOG1 N-terminal helical domain / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Brix domain / Brix domain / Brix domain profile. / Brix / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain profile. / OBG-type guanine nucleotide-binding (G) domain / OBG-type guanine nucleotide-binding (G) domain profile. / Translation initiation factor IF6 / eIF-6 family / translation initiation factor 6 / 50S ribosome-binding GTPase / Creatinase/aminopeptidase-like / GTP binding domain / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / metallochaperone-like domain / TRASH domain / Ribosomal protein L27e, conserved site / : / breast cancer carboxy-terminal domain / Ribosomal protein L34e, conserved site / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L38e protein family / Ribosomal L22e protein family / Ribosomal protein L23/L25, N-terminal / 60S ribosomal protein L35 / Ribosomal protein L35Ae, conserved site / Ribosomal protein L30e, conserved site / Ribosomal protein L34Ae / Ribosomal protein L23, N-terminal domain / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal L27e protein family / Ribosomal Protein L6, KOW domain / Ribosomal protein L30/YlxQ / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein L34e / Ribosomal protein L31e, conserved site / Ribosomal protein L37ae / Ribosomal protein L14e domain / 60S ribosomal protein L6E / Ribosomal protein L19, eukaryotic / Ribosomal protein L35A / Ribosomal protein L36e / Ribosomal protein L36e domain superfamily / Ribosomal protein L36e / Ribosomal protein L35A superfamily / Ribosomal protein L7A/L8 / Ribosomal protein L27e signature. / Ribosomal protein L32e, conserved site / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein L7, eukaryotic / Ribosomal protein L6e / Ribosomal protein L14 / Ribosomal protein L30, N-terminal
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A ...Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL2A / Large ribosomal subunit protein eL18A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Ribosome assembly protein 4 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL13A / Ribosome assembly factor MRT4 / Large ribosomal subunit protein eL14A / Ribosome biogenesis protein RPF2 / Large ribosomal subunit protein eL32 / UPF0642 protein YBL028C / Proteasome-interacting protein CIC1 / Nuclear GTP-binding protein NUG1 / Ribosome biogenesis protein NSA2 / Ribosome biogenesis protein RLP7 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / rRNA-processing protein CGR1 / Pescadillo homolog / Nucleolar GTP-binding protein 2 / Ribosome biogenesis protein 15 / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Nucleolar GTP-binding protein 1 / Probable metalloprotease ARX1 / Ribosome biogenesis protein RLP24 / Bud site selection protein 20 / Regulator of ribosome biosynthesis / Ribosome biogenesis protein NOP53 / Eukaryotic translation initiation factor 6 / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsShan W / Beril K / Kaige Y / Hailey B / Yi XZ / Dan T / Michael G / Yi Y / Zhi FL / Jelena J ...Shan W / Beril K / Kaige Y / Hailey B / Yi XZ / Dan T / Michael G / Yi Y / Zhi FL / Jelena J / Cheng YM / Jian LL / Meng QD / Woolford Jr JL / Ning G
CitationJournal: Nature / Year: 2016
Title: Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.
Authors: Shan Wu / Beril Tutuncuoglu / Kaige Yan / Hailey Brown / Yixiao Zhang / Dan Tan / Michael Gamalinda / Yi Yuan / Zhifei Li / Jelena Jakovljevic / Chengying Ma / Jianlin Lei / Meng-Qiu Dong / ...Authors: Shan Wu / Beril Tutuncuoglu / Kaige Yan / Hailey Brown / Yixiao Zhang / Dan Tan / Michael Gamalinda / Yi Yuan / Zhifei Li / Jelena Jakovljevic / Chengying Ma / Jianlin Lei / Meng-Qiu Dong / John L Woolford / Ning Gao /
Abstract: Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the ...Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
History
DepositionFeb 29, 2016-
Header (metadata) releaseApr 27, 2016-
Map releaseJun 1, 2016-
UpdateJun 15, 2016-
Current statusJun 15, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jct
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6615.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of pre-60S ribosomal subunit
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.17736803 - 0.27170941
Average (Standard dev.)-0.00018456 (±0.01024579)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1770.272-0.000

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Supplemental data

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Sample components

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Entire : state1 of pre-60S ribosome subunit

EntireName: state1 of pre-60S ribosome subunit
Components
  • Sample: state1 of pre-60S ribosome subunit
  • Complex: pre-60S ribosome

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Supramolecule #1000: state1 of pre-60S ribosome subunit

SupramoleculeName: state1 of pre-60S ribosome subunit / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: pre-60S ribosome

SupramoleculeName: pre-60S ribosome / type: complex / ID: 1 / Recombinant expression: Yes / Ribosome-details: ribosome-eukaryote: LSU 60S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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

VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy #1

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID1
DateNov 7, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID2
DateNov 22, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: OTHER / Number images used: 191848
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jct:
Cryo-em structure of eukaryotic pre-60S ribosomal subunits

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