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- EMDB-27732: eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4 -

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Basic information

Entry
Database: EMDB / ID: EMD-27732
TitleeEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4
Map dataRELION Refine3D, pixel calibrated
Sample
  • Complex: eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4
    • Complex: eEF1A(GDP)-aatRNA
    • Complex: Peptidyl-tRNA
    • Complex: Deacyl-tRNA
    • Complex: 40S subunit
    • Complex: 60S subunit
KeywordsRibosome / eEF1A / ternatin / translation
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsBrown A / Shao S / Rundlet EJ / Juette MF / Carelli JD / Taunton J / Blanchard SC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM079238 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115327-Tan United States
CitationJournal: Elife / Year: 2022
Title: Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes.
Authors: Manuel F Juette / Jordan D Carelli / Emily J Rundlet / Alan Brown / Sichen Shao / Angelica Ferguson / Michael R Wasserman / Mikael Holm / Jack Taunton / Scott C Blanchard /
Abstract: Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is ...Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.
History
DepositionJul 28, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27732.png

Downloads & links

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Map

FileDownload / File: emd_27732.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION Refine3D, pixel calibrated
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 480 pix.
= 501.6 Å		1.05 Å/pix.
x 480 pix.
= 501.6 Å		1.05 Å/pix.
x 480 pix.
= 501.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.016199289 - 0.04921464
Average (Standard dev.)-0.0000712459 (±0.0038168689)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 501.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: RELION Postprocessed, pixel calibrated

Fileemd_27732_additional_1.map
AnnotationRELION Postprocessed, pixel calibrated
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Pixel calibrated

Fileemd_27732_half_map_1.map
AnnotationPixel calibrated
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Pixel calibrated

Fileemd_27732_half_map_2.map
AnnotationPixel calibrated
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4

EntireName: eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4
Components
  • Complex: eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4
    • Complex: eEF1A(GDP)-aatRNA
    • Complex: Peptidyl-tRNA
    • Complex: Deacyl-tRNA
    • Complex: 40S subunit
    • Complex: 60S subunit

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Supramolecule #1: eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4

SupramoleculeName: eEF1A(GDP)aa-tRNA stalled on the rabbit 80S ribosome by ternatin-4
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #2: eEF1A(GDP)-aatRNA

SupramoleculeName: eEF1A(GDP)-aatRNA / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: RRL

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Supramolecule #3: Peptidyl-tRNA

SupramoleculeName: Peptidyl-tRNA / type: complex / ID: 3 / Parent: 1 / Details: P-site tRNA
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #4: Deacyl-tRNA

SupramoleculeName: Deacyl-tRNA / type: complex / ID: 4 / Parent: 1 / Details: E-site tRNA
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #5: 40S subunit

SupramoleculeName: 40S subunit / type: complex / ID: 5 / Parent: 1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #6: 60S subunit

SupramoleculeName: 60S subunit / type: complex / ID: 6 / Parent: 1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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

BufferpH: 7.4
Component:
ConcentrationNameFormula
100.0 mMPotassium acetate
5.0 mMMagnesium acetate
1.0 mMDithiothreitol (DTT)C4H10O2S2
50.0 mMHEPES (pH 7.4)C8H18N2O4S

Details: Included 1 uM terntin-4
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
DetailsPull downs of ribosomes from in vitro translation reactions of a transcript encoding 3x Flag-tagged KRas in rabbit reticulocyte lysate (RRL) in the presence of 1 uM ternatin-4.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: COUNTING / Number real images: 1422 / Average exposure time: 1.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 9.200000000000001 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 135000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 159068
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5lzs

Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 22034
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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