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- EMDB-27691: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by didemnin B -

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Basic information

Entry
Database: EMDB / ID: EMD-27691
TitleeEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by didemnin B
Map dataRelion Refine3D, pixel calibrated.
Sample
  • Complex: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by didemnin B
    • Complex: eEF1A(GDP)
    • Complex: Phe-tRNAPhe
    • Complex: Met-tRNAfMet
    • Complex: 40S subunit
    • Complex: 60S subunit
KeywordsRibosome / eEF1A / didemnin / translation
Biological speciesOryctolagus cuniculus (rabbit) / Escherichia coli (E. coli) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsRundlet EJ / Juette MF / Blanchard SC / Ferguson A / Taunton J / Carelli JD
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_27691.png

Downloads & links

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Map

FileDownload / File: emd_27691.map.gz / Format: CCP4 / Size: 244.1 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.)
1.07 Å/pix.
x 400 pix.
= 426.8 Å		1.07 Å/pix.
x 400 pix.
= 426.8 Å		1.07 Å/pix.
x 400 pix.
= 426.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.018348726 - 0.047644496
Average (Standard dev.)0.00023503571 (±0.0033179878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 426.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Relion Postprocessed, B factor -20, lowpass 4, pixel calibrated.

Fileemd_27691_additional_1.map
AnnotationRelion Postprocessed, B factor -20, lowpass 4, pixel calibrated.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Relion Refine3D, pixel calibrated.

Fileemd_27691_half_map_1.map
AnnotationRelion Refine3D, pixel calibrated.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Relion Refine3D, pixel calibrated.

Fileemd_27691_half_map_2.map
AnnotationRelion Refine3D, pixel 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 didemnin B

EntireName: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by didemnin B
Components
  • Complex: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by didemnin B
    • Complex: eEF1A(GDP)
    • Complex: Phe-tRNAPhe
    • Complex: Met-tRNAfMet
    • Complex: 40S subunit
    • Complex: 60S subunit

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

SupramoleculeName: eEF1A(GDP)aa-tRNA stalled on the human 80S ribosome by didemnin B
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 II
Details200 nM didemnin B

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 2-50 / Number grids imaged: 2 / Number real images: 3574 / Average exposure time: 10.0 sec. / Average electron dose: 67.19 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 301208
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5lzs

Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 35193
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 2 / Software - Name: RELION
FSC plot (resolution estimation)

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