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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 / homologyRibosome maturation protein SBDS, N-terminal / Ribosomal protein L10e/L16 / Ribosomal protein S5 domain 2-type fold / Ribosomal protein L6, alpha-beta domain / Ubiquitin / Ubiquitin conserved site / Ribosomal protein L3, conserved site / Ribosome maturation protein SBDS, C-terminal / Ribosomal protein L10e, conserved site / Ribosome maturation protein SBDS, conserved site ...Ribosome maturation protein SBDS, N-terminal / Ribosomal protein L10e/L16 / Ribosomal protein S5 domain 2-type fold / Ribosomal protein L6, alpha-beta domain / Ubiquitin / Ubiquitin conserved site / Ribosomal protein L3, conserved site / Ribosome maturation protein SBDS, C-terminal / Ribosomal protein L10e, conserved site / Ribosome maturation protein SBDS, conserved site / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein L11, N-terminal / Zinc-binding ribosomal protein / Translation protein, beta-barrel domain superfamily / Small GTP-binding protein domain / Translation initiation factor IF6 / Ribosomal protein L6, conserved site-2 / Ribosome maturation protein Sdo1/SBDS / Ribosomal protein L40e / Ribosomal protein L10P / Ribosomal protein L11, C-terminal / Ribosomal protein L24e, conserved site / Ribosomal protein L24e-related / Ribosomal protein L40e superfamily / 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 / Ribosomal protein L24e/L24 superfamily / Ribosome maturation protein Sdo1/SBDS, central domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Ribosomal protein L10e/L16 superfamily / Ribosomal protein L14 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 / Ribosomal protein L10e / Ribosomal protein L11/L12 / Elongation factor G C-terminus / Uncharacterized protein family UPF0023 signature. / Transcription factor, GTP-binding domain / SBDS protein C-terminal domain / Ubiquitin domain signature. / Ribosomal protein L3 signature. / Ribosomal protein L6 signature 2. / Ribosomal protein L24e signature. / Ribosomal protein L10e signature. / Ubiquitin domain profile. / Ribosomal protein L24e / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / L13a-mediated translational silencing of Ceruloplasmin expression / SRP-dependent cotranslational protein targeting to membrane / Formation of a pool of free 40S subunits / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / eIF-6 family / Ribosomal protein L11, N-terminal domain / Ribosomal protein L10 / Ribosomal protein L6 / Ribosomal protein L14P / Elongation factor EFG, domain V-like / Ribosomal protein L3 / Ribosomal L40e family / Ubiquitin domain / Shwachman-Bodian-Diamond syndrome (SBDS) protein / bone marrow development / maturation of 5.8S rRNA / inner cell mass cell proliferation / leukocyte chemotaxis / mature ribosome assembly / ribosomal subunit export from nucleus / ribosomal large subunit binding / preribosome, large subunit precursor / GTP metabolic process / bone mineralization / ribosomal large subunit biogenesis / maturation of LSU-rRNA / phagocytic vesicle / translation elongation factor activity / assembly of large subunit precursor of preribosome / translation initiation factor activity / large ribosomal subunit rRNA binding / mitotic spindle organization / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / ribosome binding / rRNA processing / spindle pole / ribosome biogenesis / ribosome / rRNA binding
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
Abstract: SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by ...SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.
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
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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 Å
α=β=γ: 90 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: UniProt:Q551M2

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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: UniProt:Q9Y3A5

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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: UniProt:Q7Z2Z2

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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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