Crystallographic Fragment Screening: Techniques & Applications

Screening a fragment library against a drug target in fragment-based drug design (FBDD) is widely used at the early stages of drug discovery projects to identify new hits that can be developed into lead molecules. While lead-like compound libraries are expected to conform to Lipinski’s rule of 5, fragments have been suggested to adhere to the rule of three. This rule specifies that the molecules in the library should have a molecular weight of less than 300 Da, fewer than three hydrogen bond donors or acceptors, fewer than three rotatable bonds, and a lipophilicity value (logP) below three.

The smaller size and fewer interactions with the target give the fragment an advantage of a higher probability of binding to the protein. However, this advantage also brings a disadvantage of low binding affinity and specificity toward the drug target. Therefore, more sensitive biophysical screening techniques, such as NMR spectroscopy, surface plasmon resonance (SPR), or thermal-shift assays, were initially employed to detect fragment binding. Crystallographic fragment screening was also introduced as an alternative screening method (REF). An X-ray structure enables the direct observation of fragment binding while allowing for the analysis of the details of its interactions with the protein. This provides a clear advantage for the subsequent steps of hit expansion and lead generation. However, the low throughput of this method and technical difficulties have limited the application of crystallographic fragment screening at the initial stage. This changed with the arrival of high-flux synchrotron beamlines, fast X-ray detectors, and the automation of many experimental steps, which substantially increased the throughput of the process and made it possible to collect hundreds of X-ray data sets per day.

These developments have significantly lowered barriers for screening larger libraries, leading to several synchrotrons now operating platforms for crystallographic fragment-based screening. Among these are the XChem facility at Diamond Light Source (UK), the Crystallographic Fragment Screening Center at Helmholtz-Zentrum Berlin at BESSY II (Germany), the screening centers at EMBL Grenoble (France), the platform at SLS (Switzerland), and, of course, the FragMAX facility at MAX IV Laboratory. Established in 2019, FragMAX is only a few kilometers from SARomics’ laboratory building. The platform offers a wide range of screening services that include various fragment libraries, laboratory automation equipment, and diverse software solutions for crystal inspection, controlling the soaking process, data collection, data reduction, and initial structure refinement.

Practical considerations
In contrast to typical protein crystallography work, which may require just a few high-quality crystals, screening a fragment library necessitates several hundred crystals for fragment soaking. As stated by the FragMax team, “the crystallization system must ensure that crystals can be grown reliably and reproducibly in large quantities without needing to prepare tens of crystallization plates.” Additionally, because library compounds are typically dissolved in DMSO, the stability of the crystals in DMSO or another organic solvent, if required, must be tested before fragment soaking. Other requirements include using 3-lens SWISSCI sitting-drop crystallization plates, crystals must be 50 μm in at least one direction, be tolerant to handling, and have a packing that allows access to the ligand binding site. Crystals with different packing (e.g., different space groups) can be beneficial, as packing variation may enhance the accessibility of the ligand-binding site and reduce the incidence of so-called false negatives. The quality and resolution of the X-ray data must also be considered, as they determine the quality of the final X-ray structure. Clearly, considering the high value of synchrotron beamtime, all these requirements must be fulfilled before arriving at the screening facility. Kanchugal et al., 2025, recently published a more detailed account of the FragMax platform. In the paper, they provide a thorough description of the platform components, including the instrumentation and software used at the different stages of the process. The paper also includes practical advice for inexperienced users and information about beamtime applications. However, our clients don’t need to apply for beamtime.

The SARomics Biostructures team has accumulated extensive experience in all forms of biophysical screening services, including crystallographic fragment screening. You can download a PDF presentation detailing our screening services or contact us directly to discuss and plan your project. All our services are operated within the framework of our agreement with MAX IV.