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- EMDB-27694: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by ternatin-4 -

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Basic information

Entry
Database: EMDB / ID: EMD-27694
TitleeEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by ternatin-4
Map datapixel calibrated
Sample
  • Complex: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by ternatin-4
    • Complex: eEF1A(GDP)
    • Complex: Phe-tRNAPhe
    • Complex: Met-tRNAfMet
    • Complex: 40S subunit
    • Complex: 60S subunitEukaryotic large ribosomal subunit (60S)
KeywordsRibosome / eEF1A / ternatin / translation
Biological speciesOryctolagus cuniculus (rabbit) / Escherichia coli (E. coli) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsRundlet EJ / Ferguson A / 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 25, 2022-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27694.png

Downloads & links

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Map

FileDownload / File: emd_27694.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpixel calibrated
Voxel sizeX=Y=Z: 1.085 Å
Density
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.021203186 - 0.056544226
Average (Standard dev.)0.00036986844 (±0.0034024515)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 434.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: RELION Postprocessed, b factor -30, lowpass 4, pixel calibrated

Fileemd_27694_additional_1.map
AnnotationRELION Postprocessed, b factor -30, lowpass 4, pixel calibrated
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: pixel calibrated

Fileemd_27694_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_27694_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 human 80S ribosome by ternatin-4

EntireName: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by ternatin-4
Components
  • Complex: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by ternatin-4
    • Complex: eEF1A(GDP)
    • Complex: Phe-tRNAPhe
    • Complex: Met-tRNAfMet
    • Complex: 40S subunit
    • Complex: 60S subunitEukaryotic large ribosomal subunit (60S)

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

SupramoleculeName: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by ternatin-4
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: eEF1A(GDP)

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

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Supramolecule #3: Phe-tRNAPhe

SupramoleculeName: Phe-tRNAPhe / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #4: Met-tRNAfMet

SupramoleculeName: Met-tRNAfMet / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #5: 40S subunit

SupramoleculeName: 40S subunit / type: complex / ID: 5 / Parent: 1
Source (natural)Organism: Homo sapiens (human) / Strain: HEK293T

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Supramolecule #6: 60S subunit

SupramoleculeName: 60S subunit / type: complex / ID: 6 / Parent: 1
Source (natural)Organism: Homo sapiens (human) / Strain: HEK293T

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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: 7.6
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
Details20 uM ternatin-4

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 2-50 / Number grids imaged: 3 / Number real images: 9401 / Average exposure time: 10.0 sec. / Average electron dose: 67.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 934291
Startup modelType of model: EMDB MAP
EMDB ID:

27691

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 2 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 34396
FSC plot (resolution estimation)

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