Multiple-step method for making exceptionally well-oriented liquid-crystalline sols of macromolecular assemblies

Area of Science:

• Biophysics
• Structural Biology
• Materials Science

Background:

• X-ray fiber diffraction is crucial for determining the atomic structure of filamentous macromolecular assemblies.
• Achieving high sample orientation is essential for resolving closely spaced layer-lines in diffraction data.
• High filament concentration is needed for orientation but is limited by high solution viscosity.

Purpose of the Study:

• To develop a systematic and reproducible method for producing highly oriented sols of filamentous macromolecules.
• To overcome the challenges posed by high viscosity in orienting concentrated macromolecular solutions.
• To enhance the capabilities of X-ray fiber diffraction for atomic structure determination.

Main Methods:

• A three-step method involving liquid crystallization, centrifugation, and magnetic orientation.
• Utilizing slow centrifugation to induce dynamic self-orientation and form homogeneous liquid-crystalline sols.
• Employing further centrifugation for concentration followed by magnetic alignment to achieve exceptional orientation.

Main Results:

• A reproducible method yielding extremely well-oriented sols was established.
• The optimized method achieved a disorientation angle of 0.6 degrees (1 sigma) in flagellar sols.
• The technique was successfully applied to various systems, including tobacco mosaic virus and F-actin.

Conclusions:

• The developed method significantly improves sample orientation for X-ray fiber diffraction.
• This advancement facilitates more accurate atomic structure determination of filamentous assemblies.
• The technique offers broad applicability across diverse biological and synthetic filamentous systems.