Ligand Screening and Discovery using Cocktail Soaking and Automated MicroED.

Area of Science:

• Structural Biology
• Biochemistry
• Crystallography

Background:

• High-throughput crystallographic screening of protein-ligand interactions traditionally uses single-crystal X-ray diffraction (SC-XRD).
• This method is limited for protein microcrystals that are too small to produce strong diffraction patterns.

Purpose of the Study:

• To develop an integrated workflow for rapid ligand screening, structure determination, and binding analysis directly from protein microcrystals.
• To overcome the limitations of SC-XRD for small microcrystals in structural biology.

Main Methods:

• Integration of cocktail soaking techniques with automated microcrystal electron diffraction (MicroED).
• Validation of the workflow using known ligands for thermolysin.
• Application to identify novel binding interactions for proteinase K ligands.

Main Results:

• Successful rapid ligand screening, structure determination, and binding analysis directly from microcrystals.
• Identification of novel binding interactions for proteinase K.
• Structures of multiple protein-ligand complexes, including those with weak binding affinities, were solved efficiently.
• Estimated relative binding affinities correlated well with previous studies and microscale thermophoresis (MST) measurements.

Conclusions:

• The developed workflow enables efficient structural analysis of protein-ligand complexes from microcrystals.
• This approach enhances the hit rate for ligand screening and provides insights into binding interactions, even for weakly binding ligands.
• The method is a valuable tool for structural biology and drug discovery efforts.