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- EMDB-40665: Puromycin reveals a distinct conformation of neuronal ribosomes. -

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Basic information

Entry
Database: EMDB / ID: EMD-40665
TitlePuromycin reveals a distinct conformation of neuronal ribosomes.
Map dataConsensus map
Sample
  • Complex: Class1_80S_with_AP_PE_tRNA
KeywordsStalled polysomes / puromycin / neuronal RNA granule / Cryo-EM / ribosome
Biological speciesRattus (rats)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsAnadolu M / Sun J / Tian-Yi-Li J / Graber T / Ortega J / Sossin WS
Funding support Canada, 1 items
OrganizationGrant numberCountry
CIFAR Azrieli Global Scholars Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Puromycin reveals a distinct conformation of neuronal ribosomes.
Authors: Mina N Anadolu / Jingyu Sun / Jewel T-Y Li / Tyson E Graber / Joaquin Ortega / Wayne S Sossin /
Abstract: Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It ...Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson , , e60048 (2020); and Enam , , e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.
History
DepositionApr 29, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40665.png

Downloads & links

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Map

FileDownload / File: emd_40665.map.gz / Format: CCP4 / Size: 240.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 398 pix.
= 433.82 Å		1.09 Å/pix.
x 398 pix.
= 433.82 Å		1.09 Å/pix.
x 398 pix.
= 433.82 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0092
Minimum - Maximum-0.021076577 - 0.04722765
Average (Standard dev.)-0.00073796127 (±0.0030012669)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions398398398
Spacing398398398
CellA=B=C: 433.82 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Multibody refine map 40S part

Fileemd_40665_additional_1.map
AnnotationMultibody refine map 40S part
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Multibody refine map 60S part

Fileemd_40665_additional_2.map
AnnotationMultibody refine map 60S part
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus half 1 map

Fileemd_40665_half_map_1.map
AnnotationConsensus half 1 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus half 2 map

Fileemd_40665_half_map_2.map
AnnotationConsensus half 2 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Class1_80S_with_AP_PE_tRNA

EntireName: Class1_80S_with_AP_PE_tRNA
Components
  • Complex: Class1_80S_with_AP_PE_tRNA

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Supramolecule #1: Class1_80S_with_AP_PE_tRNA

SupramoleculeName: Class1_80S_with_AP_PE_tRNA / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Rattus (rats)

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 615920
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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