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- EMDB-3146: Mechanism of eIF6 release from the nascent 60S ribosomal subunit -

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Entry
Database: EMDB / ID: 3146
TitleMechanism of eIF6 release from the nascent 60S ribosomal subunit
Map dataReconstruction of 60S-eIF6-SBDS-EFL1 complex
SampleDictyostelium 60S carrying endogenous eIF6 with recombinant human SBDS and human EFL1
  • ribosome-eukaryote
  • Eukaryotic translation initiation factor 6
  • Shwachman-Bodian-Diamond syndrome protein
  • Elongation factor-like 1
Keywordsribosome / ribosomopathy / SBDS / cryo-EM / eIF6 / Dictyostelium / EFL1 / GTPase / ribosome biogenesis
Function / homologyRibosomal protein L14 superfamily / Ribosomal protein L40e superfamily / Shwachman-Bodian-Diamond syndrome (SBDS) protein / Ribosomal L40e family / Elongation factor G C-terminus / Ribosomal protein L10 / Ribosomal protein L6 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L3 / Ribosomal protein L16p/L10e ...Ribosomal protein L14 superfamily / Ribosomal protein L40e superfamily / Shwachman-Bodian-Diamond syndrome (SBDS) protein / Ribosomal L40e family / Elongation factor G C-terminus / Ribosomal protein L10 / Ribosomal protein L6 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L3 / Ribosomal protein L16p/L10e / Ubiquitin family / Ribosomal protein L14p/L23e / Elongation factor Tu GTP binding domain / Ribosome maturation protein Sdo1/SBDS-like / Ribosomal protein L24e/L24 superfamily / Ribosome maturation protein Sdo1/SBDS, central domain superfamily / eIF-6 family / Ribosomal protein L10e/L16 superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L6, alpha-beta domain superfamily / Ribosome maturation protein SBDS, N-terminal domain superfamily / Ribosomal protein L11, C-terminal domain superfamily / EF-G domain III/V-like / 60S acidic ribosomal protein P0 / Ubiquitin-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Ribosomal protein L24e, conserved site / Ribosomal protein L11, N-terminal / Ribosomal protein L11, C-terminal / Ribosomal protein S5 domain 2-type fold / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L24e / Ribosomal protein L11, N-terminal domain / Ubiquitin conserved site / SRP-dependent cotranslational protein targeting to membrane / Formation of TC-NER Pre-Incision Complex / Formation of Incision Complex in GG-NER / DNA Damage Recognition in GG-NER / Metalloprotease DUBs / Ub-specific processing proteases / Josephin domain DUBs / UCH proteinases / Negative regulation of MAPK pathway / Termination of translesion DNA synthesis / Translesion synthesis by POLK / Hedgehog 'on' state / Hedgehog ligand biogenesis / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of the E3 ubiquitin ligase COP1 / APC-Cdc20 mediated degradation of Nek2A / SBDS protein C-terminal domain / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Cyclin B / L13a-mediated translational silencing of Ceruloplasmin expression / Downregulation of ERBB2:ERBB3 signaling / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ubiquitin domain profile. / Uncharacterized protein family UPF0023 signature. / Ribosomal protein L10e signature. / Ribosomal protein L24e signature. / Ribosomal protein L6 signature 2. / Ribosomal protein L3 signature. / Ubiquitin domain signature. / Ubiquitin / Ribosomal protein L3, conserved site / Gap-filling DNA repair synthesis and ligation in TC-NER / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Cyclin D associated events in G1 / Ribosome maturation protein SBDS, N-terminal / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Formation of a pool of free 40S subunits / Formation of the ternary complex, and subsequently, the 43S complex / Ribosomal scanning and start codon recognition / GTP hydrolysis and joining of the 60S ribosomal subunit / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / E3 ubiquitin ligases ubiquitinate target proteins / Ribosomal protein L14P / Regulation of PTEN localization / Regulation of PTEN stability and activity / ER Quality Control Compartment (ERQC) / Peroxisomal protein import / Endosomal Sorting Complex Required For Transport (ESCRT) / Iron uptake and transport / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Antigen processing: Ubiquitination & Proteasome degradation / Orc1 removal from chromatin / Dual incision in TC-NER / Ribosomal protein L3 / Ribosomal protein L40e / Ribosomal protein L10P / Ribosomal protein L10e / Ubiquitin domain / Ribosomal protein L24e-related / Zinc-binding ribosomal protein / Ribosomal protein S5 domain 2-type fold, subgroup / Small GTP-binding protein domain / Translation initiation factor IF6
Function and homology information
SourceDictyostelium discoideum (Slime Mold) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 4.1 Å resolution
AuthorsWeis F / Giudice E / Churcher M / Jin L / Hilcenko C / Wong CC / Traynor D / Kay RR / Warren AJ
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2015
Title: Mechanism of eIF6 release from the nascent 60S ribosomal subunit.
Authors: Félix Weis / Emmanuel Giudice / Mark Churcher / Li Jin / Christine Hilcenko / Chi C Wong / David Traynor / Robert R Kay / Alan J Warren
Validation ReportPDB-ID: 5anb

SummaryFull reportAbout validation report
DateDeposition: Sep 6, 2015 / Header (metadata) release: Sep 23, 2015 / Map release: Oct 21, 2015 / Last update: Dec 2, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5anb
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-5anb
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3146.jpg

Downloads & links

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Map

Fileemd_3146.map.gz (map file in CCP4 format, 105470 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.33 Å/pix.
= 399. Å		300 pix
1.33 Å/pix.
= 399. Å		300 pix
1.33 Å/pix.
= 399. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density

Histogram

Histogram (log scale)
Contour Level:0.06 (by author), 0.06 (movie #1):
Minimum - Maximum-0.44422188 - 0.64644915
Average (Standard dev.)0.00301982 (0.02805543)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin000
Limit299299299
Spacing300300300
CellA=B=C: 399.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z399.000399.000399.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.4440.6460.003

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Supplemental data

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Sample components

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Entire Dictyostelium 60S carrying endogenous eIF6 with recombinant human...

EntireName: Dictyostelium 60S carrying endogenous eIF6 with recombinant human SBDS and human EFL1
Number of components: 4

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Component #1: ribosome-eukaryote, 60S ribosomal subunit

Ribosome-eukaryoteName: 60S ribosomal subunit / Eukaryote: LSU 60S / Recombinant expression: No
MassTheoretical: 3 MDa / Experimental: 3 MDa
SourceSpecies: Dictyostelium discoideum (Slime Mold) / Strain: HM2917

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Component #2: protein, Eukaryotic translation initiation factor 6

ProteinName: Eukaryotic translation initiation factor 6 / a.k.a: eIF6
Details: endogenous dictyostelium protein purified bound the 60S ribosomal subunit
Recombinant expression: No / Number of Copies: 1
MassTheoretical: 26 kDa / Experimental: 26 kDa
SourceSpecies: Dictyostelium discoideum (Slime Mold) / Strain: HM2917
External referencesUniProt: Eukaryotic translation initiation factor 6

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Component #3: protein, Shwachman-Bodian-Diamond syndrome protein

ProteinName: Shwachman-Bodian-Diamond syndrome protein / a.k.a: SBDS / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 29 kDa / Experimental: 29 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pRSETA / Strain: C41
External referencesUniProt: Ribosome maturation protein SBDS

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Component #4: protein, Elongation factor-like 1

ProteinName: Elongation factor-like 1 / a.k.a: EFL1 / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 125 kDa / Experimental: 125 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: pYES2 / Strain: BCY123
External referencesUniProt: Elongation factor-like GTPase 1

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 50 mM HEPES-KOH, 100 mM K(CH3COO), 10 mM Mg(CH3COO)2, 6 mM beta-mercaptoethanol
pH: 7.5
Support filmquantifoil R2/2 glow discharged
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 100 % / Method: 6.5s blot

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Electron microscopy imaging #1

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Sep 3, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 105263 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 2200 - 2800 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Electron microscopy imaging #2

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Sep 6, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 105263 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 2200 - 2800 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3844
Details: Every image is the average of 16 frames recorded by the direct electron detector

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 11970
3D reconstructionSoftware: RELION / CTF correction: Each particle / Resolution: 4.1 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Output model

5anb

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