The analysis of a protein-ligand complex structure and the interpretation of the results depend heavily on the quality and resolution of the X-ray data, which in turn depend on the quality of the crystals used for X-ray data collection. As a rule of thumb, a resolution of 2.4 Å or better is considered best for ligand-complex characterization. It should also be noted that a substantial fraction of crystallization failures stems from poor sample quality before a crystallization drop is set. For this reason, to assess protein sample quality before running crystallization screens, we always begin with quality control (QC). The controls aim to remove factors that can prevent protein crystallization or result in poor-quality crystals and, subsequently, poor diffraction quality. Such factors include high flexibility in certain regions of the protein (typically surface loop regions), instability (and partial degradation) in the crystallization buffer at a given temperature, polydispersity (the presence of aggregated material or different oligomeric forms of the protein), and time-dependent denaturation. The importance of QC cannot be overstated and is essential to accelerate the project and prevent the loss of precious material and time in crystallization trials that yield no crystals. As a crystallization services provider, we strictly follow predefined protocols that standardize settings and procedures to ensure consistency, reproducibility, and reliability. After expressing and purifying the protein, or upon receiving it from a client, we use various biophysical techniques to assess sample quality. - We evaluate the protein’s state in solution, including aggregates, denatured material, polydispersity, and impurities. - We assess stability across various buffers, pH values, and salt concentrations commonly used in crystallization screening. - We verify the protein’s stability at room temperature and in cold storage, since crystallization trials are typically conducted at two temperatures. We use the following biophysical techniques for sample characterization: - Differential scanning fluorimetry (DSF) assesses protein stability across different buffers and temperatures. - Size-exclusion chromatography (SEC) is often required to remove potential aggregates. - Dynamic light scattering (DLS) and mass spectrometry (MS) assess sample monodispersity. - For additional quality control and to detect partially unfolded regions or regions of high flexibility, sometimes NMR spectroscopy is used via HSQC fingerprint analysis.
