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- PDB-8upt: Candidatus Methanomethylophilus alvus tRNAPyl in A-site of ribosome -

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Basic information

Entry
Database: PDB / ID: 8upt
TitleCandidatus Methanomethylophilus alvus tRNAPyl in A-site of ribosome
ComponentsRNA (71-MER)
KeywordsRNA / tRNA / pyrrolysine / translation
Function / homology: / RNA / RNA (> 10)
Function and homology information
Biological speciesCandidatus Methanomethylophilus alvus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKrahn, 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
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
Department of Energy (DOE, United States)DE-FG0298ER2031 United States
Cystic Fibrosis FoundationPUGLIS20G0 United States
Knut and Alice Wallenberg FoundationKAW 2016.0488 Sweden
CitationJournal: 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
DepositionOct 23, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (71-MER)


Theoretical massNumber of molelcules
Total (without water)22,8981
Polymers22,8981
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: RNA chain RNA (71-MER)


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

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of tRNAPyl in the A-site of the E. coli ribosome
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: in vitro transcription vector pT7-TP(deltai) (others)
Source (recombinant)Organism: in vitro transcription vector pT7-TP(deltai) (others)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Details: blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: CTFFIND / Version: 4 / Category: CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2386471 / Symmetry type: POINT

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