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- EMDB-42455: Candidatus Methanomethylophilus alvus tRNAPyl in A-site of ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-42455
TitleCandidatus Methanomethylophilus alvus tRNAPyl in A-site of ribosome
Map data
Sample
  • Complex: Complex of tRNAPyl in the A-site of the E. coli ribosome
    • RNA: RNA (71-MER)
KeywordstRNA / pyrrolysine / translation / RNA
Biological speciesin vitro transcription vector pT7-TP(deltai) (others) / Candidatus Methanomethylophilus alvus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKrahn N / Zhang J / Melnikov SV / Tharp JM / Villa A / Patel A / Howard RJ / Gabir H / Patel TR / Stetefeld J ...Krahn N / Zhang J / Melnikov SV / Tharp JM / Villa A / Patel A / Howard RJ / Gabir H / Patel TR / Stetefeld J / Puglisi J / Soll D
Funding support United States, Sweden, 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
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, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 7, 2024-
Current statusFeb 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42455.png

Downloads & links

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Map

FileDownload / File: emd_42455.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 360 pix.
= 373.32 Å		1.04 Å/pix.
x 360 pix.
= 373.32 Å		1.04 Å/pix.
x 360 pix.
= 373.32 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.037 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.153
Minimum - Maximum-0.5817245 - 1.5914505
Average (Standard dev.)0.004921247 (±0.08031818)
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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Mask #1

Fileemd_42455_msk_1.map
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Half map: #2

Fileemd_42455_half_map_1.map
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Half map: #1

Fileemd_42455_half_map_2.map
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Sample components

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Entire : Complex of tRNAPyl in the A-site of the E. coli ribosome

EntireName: Complex of tRNAPyl in the A-site of the E. coli ribosome
Components
  • Complex: Complex of tRNAPyl in the A-site of the E. coli ribosome
    • RNA: RNA (71-MER)

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Supramolecule #1: Complex of tRNAPyl in the A-site of the E. coli ribosome

SupramoleculeName: Complex of tRNAPyl in the A-site of the E. coli ribosome
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: in vitro transcription vector pT7-TP(deltai) (others)

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Macromolecule #1: RNA (71-MER)

MacromoleculeName: RNA (71-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Candidatus Methanomethylophilus alvus (archaea)
Molecular weightTheoretical: 22.897654 KDa
SequenceString:
GGGGGACGGU CCGGCGACCA GCGGGUCUCU AAAACCUAGC CAGCGGGGUU CGACGCCCCG GUCUCUCGCC A

GENBANK: GENBANK: LR699000.1

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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: 7.4
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5ud5


Details: used the Mazei tRNA Pyl from 5UD5
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2386471
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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