Instrumentation and methods for efficient time-resolved X-ray crystallography of biomolecular systems with sub-10 ms...

Area of Science:

• Structural Biology
• Biochemistry
• Crystallography

Background:

• Time-resolved X-ray crystallography elucidates enzyme mechanisms at atomic resolution.
• Current methods demand complex beamline instrumentation and vast crystal quantities.
• Significant technical hurdles limit routine application and high-throughput screening.

Purpose of the Study:

• To develop simplified, high-throughput instrumentation for time-resolved X-ray crystallography.
• To enable millisecond-timescale structural studies of biomolecular reactions.
• To facilitate routine laboratory use and mail-in data collection at synchrotron beamlines.

Main Methods:

• Developed novel instrumentation for rapid reaction initiation via crystal-substrate mixing.
• Employed thermal quenching for swift structural state capture without pre-cooling.
• Utilized standard crystallography sample supports for compatibility with high-throughput cryocrystallography beamlines.

Main Results:

• Achieved time resolutions in the single-millisecond range, comparable to advanced techniques.
• Demonstrated high-throughput capability using standard sample supports and mail-in data collection.
• Determined structures of N-acetylglucosamine binding to lysozyme from 8 ms to 2 s using one crystal per time point.

Conclusions:

• The new approach offers a simple, robust, and cost-effective method for time-resolved structural studies.
• This technique is adaptable for diverse ligand solutions and requires minimal sample volumes.
• The methodology is well-suited for routine laboratory use and high-throughput screening of biomolecular systems.