To streamline and maximize the speed and efficiency of our services, our staff follows specific protocols throughout all crystallization and protein X-ray structure determination experiments. To ensure the quality of client samples, we have also developed general guidelines that you can download for your convenience. The process of protein structure determination by X-ray crystallography includes five main stages: - Crystallization (initial screening and crystal hit optimization) - Crystal harvesting, testing, and X-ray data collection - Obtaining phases, calculating the electron density map, and building the structural model - Refinement and overall quality assessment against Protein Data Bank (PDB) criteria - In the case of a ligand complex, we need to apply specific criteria to ligand-protein complex structure validation and quality assessment At SARomics Biostructures, both crystal testing and diffraction data collection are primarily conducted at the protein crystallography beamlines BioMax and MicroMax at the 4th-generation synchrotron MAX IV Laboratory in Lund. For data collection and processing, we use the crystallographic software packages XDS and autoPROC (Global Phasing). With a synchrotron nearby, we enjoy quick access to beamlines, eliminating the need to ship crystals over long distances to remote synchrotrons and the risk of crystal damage during transport. Generally, to minimize radiation damage from modern high-intensity synchrotron beams, data collection must be conducted at cryogenic temperatures. To this end, after harvesting from drops, crystals are transferred to a cryo-solution and flash-frozen at liquid nitrogen temperature. The cryo-solution prevents ice formation around a crystal when it is mounted in a special loop before insertion into an X-ray beam. Ice interferes with data collection (ice crystals scatter X-rays) and may also damage the protein crystals, resulting in poor data quality. Crystallographic software packages Phaser and Molrep are typically used to phase macromolecular crystal structures using molecular replacement and single-wavelength anomalous dispersion. If molecular replacement fails, we must use de novo structure determination. For this, typically, a selenomethionine (SeMet)-labeled protein sample, which we can produce upon the client's request, enables structure determination using synchrotron radiation and anomalous X-ray scattering. Crystallographic software such as REFMAC (CCP4) and BUSTER (Global Phasing), along with the graphics program Coot (CCP4), are used for model building and refinement.
