Room-temperature X-ray fragment screening with serial crystallography

Area of Science:

• Structural biology
• Drug discovery
• Biochemistry

Background:

• Macromolecular X-ray crystallography is key for understanding protein-ligand interactions in drug development.
• Cryogenic temperature data collection, while standard, can introduce artifacts affecting protein conformation and interactions.
• Room-temperature (RT) crystallography offers a more physiologically relevant approach.

Purpose of the Study:

• To compare RT fragment screening using advanced fixed-target serial crystallography with conventional cryogenic methods.
• To investigate the impact of temperature on protein-ligand interactions and identify novel binding sites.
• To assess the potential of RT serial crystallography for high-throughput fragment screening.

Main Methods:

• Fixed-target serial crystallography at room temperature (RT).
• Conventional single crystal X-ray crystallography at cryogenic temperatures (cryo).
• Systematic fragment screening of Fosfomycin-resistance protein A (FosAKP) from Klebsiella pneumoniae.

Main Results:

• RT serial crystallography achieved resolutions comparable to cryo methods.
• A previously unobserved active site conformational state of FosAKP was identified at RT.
• Ligand binding modes were consistent across temperatures, but more binders were observed at cryo.

Conclusions:

• RT serial crystallography is a viable high-throughput method for fragment screening.
• This technique can uncover novel protein conformations relevant for drug design.
• RT crystallography provides valuable structural insights closer to physiological conditions.