Journal Article

Room-temperature X-ray fragment screening with serial crystallography

Thekku Veedu, Sreevidya, Rodrigues, Ana Carolina, Falke, Sven, Reinke, Patrick Y. A., Galchenkova, Marina, Fischer, Pontus, Chapman, Henry N., Elinjikkal, Daniel, Senst, Johanna, Meyer, Jan, Barthelmess, Miriam, Meents, Alke, Günther, Sebastian, Gumprecht, Lars

2025

Overview

Structural insights into protein-ligand interactions are essential for advancing drug development, with macromolecular X-ray crystallography being a cornerstone technique. Commonly X-ray data collection is conducted at cryogenic temperatures to mitigate radiation damage effects. However, this can introduce artifacts not only in the protein conformation but also in protein-ligand interactions. Recent studies highlight the advantages of room-temperature (RT) crystallography in capturing relevant states much closer to physiological temperatures. We have advanced fixed-target serial crystallography to enable high-throughput fragment screening at RT. Here we systematically compare RT fragment screening of the Fosfomycin-resistance protein A from Klebsiella pneumoniae (FosAKP), an enzyme involved in antibiotic resistance, with conventional single crystal data collection at cryogenic temperature (cryo). With RT serial crystallography we achieve resolutions comparable to cryogenic methods and identify a previously unobserved conformational state of the active site, offering additional starting points for drug design. For ligands identified in both screens, temperature does not have an influence on the binding mode of the ligand. But overall, we observe more binders at cryo, both at physiologically relevant and non-relevant sites. With the potential for further automation, RT screening with serial crystallography can advance drug development pipelines by making undiscovered conformations of proteins accessible. The authors in this work apply room-temperature serial X-ray crystallography to fragment screening. This reveals distinct protein conformations and altered binding modes when compared to conventional cryogenic methods, whilst providing similar resolution.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 101/47 / 631/154/1435/2163 / 631/1647/2258/1266 / 631/535/1266 / Antibiotic resistance / Automation / Bacterial Proteins - chemistry / Bacterial Proteins - metabolism / Binders / Binding / Catalytic Domain / Cryogenic temperature / Crystallization / Crystallography / Crystallography, X-Ray - methods / Data collection / Dehydration / Drug development / Enzymes / Experiments / Fosfomycin / Humanities and Social Sciences / Humidity / Klebsiella / Klebsiella pneumoniae - enzymology / Libraries / Ligands / Microscopy / Models, Molecular / multidisciplinary / Protein A / Protein Binding / Protein Conformation / Protein structure / Proteins / Radiation / Radiation damage / Reproducibility / Room temperature / Science / Science (multidisciplinary) / Screening / Single crystals / Temperature / X-ray crystallography