9O2X

Structure of WT E.coli ribosome 70S subunit with complexed with mRNA, P-site fMet-NH-tRNAfMet and A-site (S)-betahydroxyBocK charged NH-tRNAPyl

This is a non-PDB format compatible entry.

Summary for 9O2X

• Entry DOI: 10.2210/pdb9o2x/pdb
• EMDB information: 70050
• Descriptor: 50S ribosomal protein L33, Small ribosomal subunit protein uS7, Small ribosomal subunit protein uS8, ... (64 entities in total)
• Functional Keywords: ribosome, 70s, unnatural amino acids
• Biological source: Escherichia coli; More
• Total number of polymer chains: 55
• Total formula weight: 2208959.85

Authors

Majumdar, C.,Kent, A.,Hamlish, N.,Zhu, C.,Cate, J. (deposition date: 2025-04-04, release date: 2026-03-18)

Primary citation

Majumdar, C.,Kent, A.D.,Hamlish, N.X.,Zhu, C.,Fitzgerald, K.A.,Cate, J.H.D.,Schepartz, A.
Co-Translational Incorporation of (R) - and (S) -beta 2 -Hydroxy Acids In Vitro : A Structural and Biochemical Study on the E. coli Ribosome.
J.Am.Chem.Soc., 2026

PubMed Abstract: Engineering the translation apparatus to accept backbone-modified amino acid analogues would enable the programmed synthesis of sequence-defined biopolymers with tunable properties. β-Hydroxy acids are of particular interest because they could support the programmed biosynthesis of both biocompatible polyester materials as well as natural product-like depsipeptides. Previous work has reported that both enantiomers of β-hydroxy-N-Boc-lysine (β-OH-BocK) are substrates for the orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair, but only one enantiomer is introduced into protein . Here we make use of high-resolution cryogenic electron microscopy (cryo-EM) to determine the structural basis for this observation. These structures reveal both β-OH-BocK isomers equally well-positioned within the ribosomal A site regardless of stereochemistry. Consistent with this observation, translation reactions charged with tRNAs acylated with - or -β-OH-BocK produced roughly equal amounts of translated product when quantified on the basis of either mass spectrometry or luminescence. Together, these experiments imply that the substantial preferential incorporation of one enantiomer over the other observed previously results primarily from deficiencies in the steps that precede bond formation by the ribosome. Indeed, as predicted by this work and demonstrated in an accompanying paper (Soni, C. Co-Translational Incorporation of ()- and ()-β-Hydroxyacids : Directed Evolution of Efficient Aminoacyl-tRNA Synthetases. 2026, 148, 10.1021/jacs.5c18595), when cells are provided with an active and orthogonal aminoacyl-tRNA synthetase/tRNA pair that accepts both - and -β-OH-BocK as substrates, both monomers are introduced into protein in good yield and with high fidelity.

PubMed: 41757712
DOI: 10.1021/jacs.5c18603

Experimental method

ELECTRON MICROSCOPY (2.29 Å)