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- EMDB-26713: allo-tRNAUTu1A in the A site of the E. coli ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-26713
Titleallo-tRNAUTu1A in the A site of the E. coli ribosome
Map dataPrimary Map
Sample
  • Complex: E. coli 70S ribosome with allo-tRNAUTu1A in the A site
    • RNA: Allo-tRNAUTu1A
Biological speciesEscherichia coli (E. coli) / metagenome (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsZhang J / Prabhakar A / Krahn N / Vargas-Rodriguez O / Krupkin M / Fu Z / Acosta-Reyes FJ / Ge X / Choi J / Crnkovic A ...Zhang J / Prabhakar A / Krahn N / Vargas-Rodriguez O / Krupkin M / Fu Z / Acosta-Reyes FJ / Ge X / Choi J / Crnkovic A / Ehrenberg M / Viani Puglisi E / Soll D / Puglisi J
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM122560 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM122560-05S1 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM51266 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI15046 United States
Department of Energy (DOE, United States)DE-FG0298ER2031 United States
Cystic Fibrosis FoundationPUGLIS20G0 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Uncovering translation roadblocks during the development of a synthetic tRNA.
Authors: Arjun Prabhakar / Natalie Krahn / Jingji Zhang / Oscar Vargas-Rodriguez / Miri Krupkin / Ziao Fu / Francisco J Acosta-Reyes / Xueliang Ge / Junhong Choi / Ana Crnković / Måns Ehrenberg / ...Authors: Arjun Prabhakar / Natalie Krahn / Jingji Zhang / Oscar Vargas-Rodriguez / Miri Krupkin / Ziao Fu / Francisco J Acosta-Reyes / Xueliang Ge / Junhong Choi / Ana Crnković / Måns Ehrenberg / Elisabetta Viani Puglisi / Dieter Söll / Joseph Puglisi /
Abstract: Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ...Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
History
DepositionApr 22, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26713.png

Downloads & links

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Map

FileDownload / File: emd_26713.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary Map
Voxel sizeX=Y=Z: 1.037 Å
Density
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-4.493112 - 7.8817124
Average (Standard dev.)0.0013481326 (±0.23962839)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 373.31998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_26713_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_26713_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli 70S ribosome with allo-tRNAUTu1A in the A site

EntireName: E. coli 70S ribosome with allo-tRNAUTu1A in the A site
Components
  • Complex: E. coli 70S ribosome with allo-tRNAUTu1A in the A site
    • RNA: Allo-tRNAUTu1A

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Supramolecule #1: E. coli 70S ribosome with allo-tRNAUTu1A in the A site

SupramoleculeName: E. coli 70S ribosome with allo-tRNAUTu1A in the A site
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Allo-tRNAUTu1A

MacromoleculeName: Allo-tRNAUTu1A / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 28.782008 KDa
SequenceString:
GGAGGGGAAA UUCUAUCUGG UGAUAGACGG GAACUCUAAA UUCCUUGAAA UGCCUCGCCG CAUUGGGUUC GAUUCCCUUC CCCUCCGCC A

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV / Details: blot for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10850093
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 424828

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