Ribosome engineering to promote new crystal forms.

Area of Science:

• Molecular biology
• Structural biology
• Biochemistry

Background:

• Crystallographic studies have elucidated ribosome structure and protein synthesis.
• Crystallizing functional ribosome-GTPase translation factor complexes remained a challenge for a decade.
• Crystal packing often interferes with the binding of GTPase factors to the 16S rRNA.

Purpose of the Study:

• To overcome the crystallization challenges of ribosome-GTPase factor complexes.
• To obtain high-resolution structures of ribosome complexes with translation factors.
• To facilitate structural studies of protein synthesis.

Main Methods:

• Analysis of crystal packing in different 70S ribosome forms.
• Identification of a specific crystal contact involving ribosomal protein L9 and 16S rRNA.
• Construction of a mutant Thermus thermophilus strain (HB8-MRCMSAW1) with a truncated ribosomal protein L9 gene.
• Homologous recombination for strain construction.
• Crystallization and structure determination of ribosome-EF-G-GDP-fusidic acid complex using mutant ribosomes.

Main Results:

• A crystal packing contact between ribosomal protein L9 and 16S rRNA was identified as a major obstacle.
• A mutant Thermus thermophilus strain lacking the L9 gene interaction was successfully constructed.
• The mutant ribosomes enabled the crystallization and structural determination of the ribosome-EF-G-GDP-fusidic acid complex in a novel crystal form.
• This mutant strain proved useful for crystallizing other ribosome-GTPase factor complexes.

Conclusions:

• Truncating the ribosomal protein L9 gene effectively prevents the formation of inhibitory crystal contacts.
• The developed mutant strain is a valuable tool for structural studies of ribosome complexes with GTPase translation factors.
• This work significantly advances the ability to obtain high-resolution structures of key protein synthesis machinery.