A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Mounting microcrystals (<10 µm) for single crystal cryo-crystallography is challenging.
• Improvements in X-ray diffraction data quality depend on sample environment and preparation.
• Reducing background noise is crucial for analyzing weak diffraction from microcrystals.

Purpose of the Study:

• To present a protocol for preparing and assessing soluble protein microcrystals for X-ray diffraction.
• To detail methods for reducing X-ray background noise during microcrystal analysis.
• To demonstrate sample preparation techniques adapted from cryo-electron microscopy for the VMXm beamline.

Main Methods:

• Utilized an in-vacuum sample environment at the VMXm beamline to eliminate air path noise.
• Adapted cryo-electron microscopy sample preparation techniques, including copper grids with holey carbon films.
• Employed automated blotting and plunge cooling in liquid ethane for rapid sample freezing.
• Assessed microcrystal quality using visible light and scanning electron microscopy prior to X-ray diffraction.

Main Results:

• Developed protocols enabling the preparation of hundreds of microcrystals on a single grid with minimal surrounding liquid.
• Minimized crystalline ice formation through rapid plunge cooling.
• Demonstrated reduction of major X-ray background noise sources.
• Presented examples of successful and optimized microcrystal sample preparations.

Conclusions:

• The integrated approach of advanced beamline instrumentation and cryo-electron microscopy-adapted sample preparation significantly improves microcrystal mounting and data quality for X-ray crystallography.
• This methodology provides a robust framework for preparing and analyzing microcrystals, crucial for structural determination of challenging protein targets.
• Further optimization strategies are available for samples requiring additional refinement.