In situ counter-diffusion crystallization and long-term crystal preservation in microfluidic fixed targets for serial...

Area of Science:

• Crystallography
• Structural Biology
• Materials Science

Background:

• Counter diffusion crystallization offers advantages over batch and vapor diffusion methods for protein crystal growth.
• Current fixed-target devices for counter diffusion crystallization are limited.
• Existing methods often require complex crystal handling for X-ray diffraction.

Purpose of the Study:

• To present user-friendly designs for counter diffusion chambers integrated into 2D polymer microfluidic fixed-target chips.
• To detail methods for rapid fabrication of these microfluidic chips using common thin-film materials.
• To provide guidelines for optimizing nucleation and crystal growth while minimizing sample consumption.

Main Methods:

• Fabrication of 2D polymer microfluidic chips using Mylar, propylene, and Kapton thin films.
• Development of user-friendly counter diffusion chambers within the microfluidic chips.
• Optimization of crystallization conditions for nucleation and growth.
• Direct X-ray diffraction analysis of crystals within the microfluidic chip.

Main Results:

• Successful growth of large protein crystals with maintained hydration for extended periods.
• Demonstration of direct use of the microfluidic chip at X-ray beamlines, preserving crystal quality.
• Achieved 1.32 Å resolution diffraction data for photoactive yellow protein at room temperature.
• Simplified chip fabrication and enhanced stability under vacuum.

Conclusions:

• The presented microfluidic fixed-target chip designs provide a reliable method for growing large protein crystals.
• This approach simplifies crystal handling, reduces background scatter, and preserves crystal quality for X-ray diffraction.
• The advances in microfluidic fixed-target technology are expected to increase its adoption in crystallography.