Approaching the molecular structure of ribosomes

Area of Science:

• Structural biology
• Molecular biology
• Biochemistry

Background:

• Ribosomal particles are essential for protein biosynthesis.
• Obtaining high-resolution structural data of ribosomes is challenging due to their complexity and sensitivity.

Purpose of the Study:

• To develop methods for crystallizing biologically active ribosomal particles.
• To determine the high-resolution structure of ribosomal subunits and the complete ribosome.
• To gain insights into the mechanism of protein biosynthesis.

Main Methods:

• Crystallization of ribosomal particles in three-dimensional and two-dimensional forms.
• Cryo-crystallography using synchrotron radiation at low temperatures.
• Heavy-atom cluster soaking and specific binding for phasing.
• Isomorphous crystallization of mutant Bacillus stearothermophilus 50 S ribosomal subunits.
• Reconstruction of 70 S ribosome and 50 S subunit models from 2D sheets.

Main Results:

• Successfully crystallized 15 forms of 3D crystals and 3 forms of 2D sheets from ribosomal particles.
• Crystallized particles retained biological activity for extended periods.
• Collected complete diffraction data to ~6 Å resolution from a single crystal using cryo-conditions with minimal radiation damage.
• Determined structures of the 70 S ribosome (47 Å) and 50 S subunit (30 Å).
• Identified key structural features including subunit interfaces, potential protein biosynthesis sites, and a polypeptide exit tunnel.

Conclusions:

• Cryo-crystallography is a viable method for high-resolution ribosome structure determination.
• The determined structures provide a detailed map of the ribosome, elucidating its functional architecture.
• Structural insights support models of protein biosynthesis and polypeptide translocation through the ribosome.