Cryo2RT: a high-throughput method for room-temperature macromolecular crystallography from cryo-cooled crystals

Area of Science:

• Structural biology
• Biophysics
• Drug discovery

Background:

• Synchrotron cryo-crystallography and cryo-electron microscopy are vital for structural biology and drug discovery.
• Discrepancies between cryogenic and room-temperature crystal structures present significant challenges in understanding biological systems and developing therapeutics.

Purpose of the Study:

• To introduce Cryo2RT, a novel high-throughput method for room-temperature (RT) data collection from cryo-cooled crystals.
• To address the limitations posed by structural disparities between cryogenic and RT states.
• To facilitate the study of structural dynamics and ligand-binding at physiological temperatures.

Main Methods:

• Cryo2RT utilizes the established cryo-crystallography workflow for data collection.
• The method was tested on endothiapepsin crystals soaked with four fragments.
• Additional tests were performed on thaumatin and SARS-CoV-2 3CLpro.

Main Results:

• Cryo2RT successfully collected RT data from cryo-cooled crystals.
• Unique ligand-binding poses were identified, differing from those observed at cryogenic temperatures.
• The method demonstrated throughput comparable to traditional cryo-crystallography.
• Exploration of structural dynamics at various temperatures was facilitated.

Conclusions:

• Cryo2RT provides a high-throughput solution for obtaining room-temperature structural data from cryo-cooled crystals.
• The method overcomes challenges associated with cryogenic-vs-RT structural discrepancies.
• Cryo2RT enhances the study of biological processes and drug discovery by enabling analysis at physiologically relevant temperatures.