Fixed-target protein serial microcrystallography with an x-ray free electron laser

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• Serial femtosecond crystallography (SFX) is crucial for determining protein structures.
• Traditional SFX methods often require large sample quantities and can be limited by radiation damage.
• Developing methods for high-resolution structure determination with minimal sample consumption is essential.

Purpose of the Study:

• To demonstrate high-resolution SFX (~2.5 Å) using protein microcrystals on silicon nitride membranes.
• To evaluate the feasibility of room-temperature data acquisition with reduced sample usage.
• To assess the potential for high-throughput data collection using X-ray Free Electron Laser (XFEL) sources.

Main Methods:

• Utilized protein microcrystals embedded in a preservation medium on an ultra-thin silicon nitride membrane.
• Employed X-ray Free Electron Laser (XFEL) sources for high-speed data acquisition (up to 10 Hz).
• Applied the diffraction-before-destruction technique to mitigate radiation damage.

Main Results:

• Achieved high-resolution diffraction data (~2.5 Å) at room temperature.
• Demonstrated a peak data acquisition rate of 10 Hz with a ~38% hit rate.
• Significantly reduced sample consumption compared to traditional flowing jet methods.

Conclusions:

• This membrane-based SFX approach enables ultra-low sample consumption for high-resolution structure determination.
• The method is suitable for proteins available in small quantities or those difficult to crystallize in bulk.
• This technique opens new avenues for studying challenging protein targets using XFELs.