+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26714 | |||||||||||||||||||||
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Title | allo-tRNAUTu1A in the P site of the E. coli ribosome | |||||||||||||||||||||
Map data | Primary Map | |||||||||||||||||||||
Sample |
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Keywords | tRNA / selenocysteine / ribosome / RNA | |||||||||||||||||||||
Biological species | Escherichia coli (E. coli) / metagenome (others) | |||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||||||||||||||
Authors | Zhang 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
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26714.map.gz | 168.2 MB | EMDB map data format | |
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Header (meta data) | emd-26714-v30.xml emd-26714.xml | 15.2 KB 15.2 KB | Display Display | EMDB header |
Images | emd_26714.png | 51.2 KB | ||
Filedesc metadata | emd-26714.cif.gz | 4.7 KB | ||
Others | emd_26714_half_map_1.map.gz emd_26714_half_map_2.map.gz | 165 MB 165 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26714 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26714 | HTTPS FTP |
-Validation report
Summary document | emd_26714_validation.pdf.gz | 1.3 MB | Display | EMDB validaton report |
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Full document | emd_26714_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | emd_26714_validation.xml.gz | 15.2 KB | Display | |
Data in CIF | emd_26714_validation.cif.gz | 18 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26714 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26714 | HTTPS FTP |
-Related structure data
Related structure data | 7urmMC 7ur5C 7uriC M: atomic model generated by this map C: citing same article (ref.) |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_26714.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Primary Map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.037 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half Map 1
File | emd_26714_half_map_1.map | ||||||||||||
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Annotation | Half Map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map 2
File | emd_26714_half_map_2.map | ||||||||||||
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Annotation | Half Map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : E. coli 70S ribosome with allo-tRNAUTu1A in the P site
Entire | Name: E. coli 70S ribosome with allo-tRNAUTu1A in the P site |
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Components |
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-Supramolecule #1: E. coli 70S ribosome with allo-tRNAUTu1A in the P site
Supramolecule | Name: E. coli 70S ribosome with allo-tRNAUTu1A in the P site type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: allo-tRNAUTU1A
Macromolecule | Name: allo-tRNAUTU1A / type: rna / ID: 1 / Number of copies: 1 |
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Source (natural) | Organism: metagenome (others) |
Molecular weight | Theoretical: 28.782008 KDa |
Sequence | String: GGAGGGGAAA UUCUAUCUGG UGAUAGACGG GAACUCUAAA UUCCUUGAAA UGCCUCGCCG CAUUGGGUUC GAUUCCCUUC CCCUCCGCC A |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | 3D array |
-Sample preparation
Buffer | pH: 7 |
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Vitrification | Cryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV / Details: blot for 3 seconds before plunging. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |