+Open data
-Basic information
Entry | Database: PDB / ID: 7urm | |||||||||||||||||||||
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Title | allo-tRNAUTu1A in the P site of the E. coli ribosome | |||||||||||||||||||||
Components | allo-tRNAUTU1A | |||||||||||||||||||||
Keywords | RNA / tRNA / selenocysteine / ribosome | |||||||||||||||||||||
Function / homology | RNA / RNA (> 10) Function and homology information | |||||||||||||||||||||
Biological species | metagenome (others) | |||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | |||||||||||||||||||||
Authors | Zhang, J. / Prabhakar, A. / Krahn, N. / Vargas-Rodriguez, O. / Krupkin, M. / Fu, Z. / Acosta-Reyes, F.J. / Ge, X. / Choi, J. / Crnkovic, A. ...Zhang, J. / Prabhakar, A. / Krahn, N. / Vargas-Rodriguez, O. / Krupkin, M. / Fu, Z. / Acosta-Reyes, F.J. / Ge, X. / Choi, J. / Crnkovic, A. / Ehrenberg, M. / Viani Puglisi, E. / Soll, D. / Puglisi, J. | |||||||||||||||||||||
Funding support | United States, 6items
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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
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7urm.cif.gz | 56.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7urm.ent.gz | 40.7 KB | Display | PDB format |
PDBx/mmJSON format | 7urm.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7urm_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7urm_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7urm_validation.xml.gz | 18.5 KB | Display | |
Data in CIF | 7urm_validation.cif.gz | 23.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ur/7urm ftp://data.pdbj.org/pub/pdb/validation_reports/ur/7urm | HTTPS FTP |
-Related structure data
Related structure data | 26714MC 7ur5C 7uriC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: RNA chain | Mass: 28782.008 Da / Num. of mol.: 1 / Source method: obtained synthetically Details: Oil polluted marine microbial communities from Coal Oil Point, Santa Barbara, California, USA - Santa Barbara Oil Seep Sample 6 (Crude oil metagenome 6, ASSEMBLY_DATE=20131204) Source: (synth.) metagenome (others) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: E. coli 70S ribosome with allo-tRNAUTu1A in the P site Type: RIBOSOME / Entity ID: all / Source: NATURAL |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Details: blot for 3 seconds before plunging |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software | Name: CTFFIND / Version: 4 / Category: CTF correction |
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CTF correction | Type: NONE |
Particle selection | Num. of particles selected: 10850093 |
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23609 / Symmetry type: POINT |