Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Time-resolved serial crystallography is crucial for observing dynamic biological processes.
• Existing methods face challenges in precision and data consistency across different facilities.

Purpose of the Study:

• To develop and implement a novel drop-on-fixed-target method for time-resolved serial crystallography.
• To enhance the accuracy and reliability of crystallographic studies at synchrotron and X-ray free-electron laser (XFEL) facilities.

Main Methods:

• Utilized a piezoelectric droplet dispensing pipette for precise picoliter droplet delivery.
• Developed a high-density crystalline silicon fixed-target chip with 25,600 wells for enzyme microcrystals.
• Implemented advanced motion controls and strategies to mitigate cross-well contamination.

Main Results:

• Successfully tested the system with lysozyme and two beta-lactamases (CTX-M-15 and AmpC_EC).
• Demonstrated robust performance and data acquisition at both synchrotron and XFEL sources.
• Validated the effectiveness of interleaved controls for managing cross-well contamination.

Conclusions:

• The drop-on-fixed-target method provides a reliable framework for time-resolved crystallography.
• This technique significantly improves the consistency and accuracy of measurements across diverse experimental facilities.
• Facilitates more robust time-resolved crystallographic studies of enzyme dynamics.