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Title: Ribosome engineering to promote new crystal forms

Abstract

Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

Authors:
 [1]; ;
  1. Uppsala University, Box 596, SE-751 24 Uppsala (Sweden)
Publication Date:
OSTI Identifier:
22347885
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section D: Biological Crystallography
Additional Journal Information:
Journal Volume: 68; Journal Issue: Pt 5; Other Information: PMCID: PMC3335287; PMID: 22525755; PUBLISHER-ID: xb5044; OAI: oai:pubmedcentral.nih.gov:3335287; Copyright (c) Selmer et al. 2012; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0907-4449
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTAL LATTICES; CRYSTALLIZATION; CRYSTALS; ELONGATION; ENGINEERING; PROTEINS; RECOMBINATION; SPACE GROUPS; STOWING; STRAINS; SYNTHESIS

Citation Formats

Selmer, Maria, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, Gao, Yong-Gui, Weixlbaumer, Albert, Ramakrishnan, V., E-mail: maria.selmer@icm.uu.se, and Uppsala University, Box 596, SE-751 24 Uppsala. Ribosome engineering to promote new crystal forms. Denmark: N. p., 2012. Web. doi:10.1107/S0907444912006348.
Selmer, Maria, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, Gao, Yong-Gui, Weixlbaumer, Albert, Ramakrishnan, V., E-mail: maria.selmer@icm.uu.se, & Uppsala University, Box 596, SE-751 24 Uppsala. Ribosome engineering to promote new crystal forms. Denmark. https://doi.org/10.1107/S0907444912006348
Selmer, Maria, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, Gao, Yong-Gui, Weixlbaumer, Albert, Ramakrishnan, V., E-mail: maria.selmer@icm.uu.se, and Uppsala University, Box 596, SE-751 24 Uppsala. 2012. "Ribosome engineering to promote new crystal forms". Denmark. https://doi.org/10.1107/S0907444912006348.
@article{osti_22347885,
title = {Ribosome engineering to promote new crystal forms},
author = {Selmer, Maria and MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH and Gao, Yong-Gui and Weixlbaumer, Albert and Ramakrishnan, V., E-mail: maria.selmer@icm.uu.se and Uppsala University, Box 596, SE-751 24 Uppsala},
abstractNote = {Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.},
doi = {10.1107/S0907444912006348},
url = {https://www.osti.gov/biblio/22347885}, journal = {Acta Crystallographica. Section D: Biological Crystallography},
issn = {0907-4449},
number = Pt 5,
volume = 68,
place = {Denmark},
year = {Tue May 01 00:00:00 EDT 2012},
month = {Tue May 01 00:00:00 EDT 2012}
}