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Yorodumi- PDB-8upt: Candidatus Methanomethylophilus alvus tRNAPyl in A-site of ribosome -
+Open data
-Basic information
Entry | Database: PDB / ID: 8upt | |||||||||||||||||||||
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Title | Candidatus Methanomethylophilus alvus tRNAPyl in A-site of ribosome | |||||||||||||||||||||
Components | RNA (71-MER) | |||||||||||||||||||||
Keywords | RNA / tRNA / pyrrolysine / translation | |||||||||||||||||||||
Function / homology | : / RNA / RNA (> 10) Function and homology information | |||||||||||||||||||||
Biological species | Candidatus Methanomethylophilus alvus (archaea) | |||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å | |||||||||||||||||||||
Authors | Krahn, N. / Zhang, J. / Melnikov, S.V. / Tharp, J.M. / Villa, A. / Patel, A. / Howard, R.J. / Gabir, H. / Patel, T.R. / Stetefeld, J. ...Krahn, N. / Zhang, J. / Melnikov, S.V. / Tharp, J.M. / Villa, A. / Patel, A. / Howard, R.J. / Gabir, H. / Patel, T.R. / Stetefeld, J. / Puglisi, J. / Soll, D. | |||||||||||||||||||||
Funding support | United States, Sweden, 6items
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Citation | Journal: Nucleic Acids Res / Year: 2024 Title: tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut. Authors: Natalie Krahn / Jingji Zhang / Sergey V Melnikov / Jeffery M Tharp / Alessandra Villa / Armaan Patel / Rebecca J Howard / Haben Gabir / Trushar R Patel / Jörg Stetefeld / Joseph Puglisi / Dieter Söll / Abstract: Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction ...Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68). | |||||||||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8upt.cif.gz | 45.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8upt.ent.gz | 32 KB | Display | PDB format |
PDBx/mmJSON format | 8upt.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8upt_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 8upt_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 8upt_validation.xml.gz | 26.8 KB | Display | |
Data in CIF | 8upt_validation.cif.gz | 36.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/up/8upt ftp://data.pdbj.org/pub/pdb/validation_reports/up/8upt | HTTPS FTP |
-Related structure data
Related structure data | 42455MC M: map data used to model this data C: citing same article (ref.) |
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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: 22897.654 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Candidatus Methanomethylophilus alvus (archaea) Gene: pylT Production host: in vitro transcription vector pT7-TP(deltai) (others) References: GenBank: 1721134198 |
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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: Complex of tRNAPyl in the A-site of the E. coli ribosome Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: in vitro transcription vector pT7-TP(deltai) (others) |
Source (recombinant) | Organism: in vitro transcription vector pT7-TP(deltai) (others) |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 |
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: PHASE FLIPPING AND AMPLITUDE CORRECTION |
3D reconstruction | Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2386471 / Symmetry type: POINT |