Crystallization experiments are set up for initial screening of crystallization conditions. Once initial crystals are obtained and confirmed as the crystals of the target macromolecule using crystal dye and/or X-ray diffraction, the crystallization conditions are optimized in house to generate seeds for growth of single crystal.

For initial screening, the client will provide the following information and materials if proteins of interest are not prepared by Creative BioStructure:

Sample Information:

o    Purity of the protein by SDS PAGE and/or Mass Spectrometry. We recommend that the purity of target protein should be at least 90% to 95% based on a Coomassie-stained SDS-PAGE. Creative BioStructure can purify submitted sample to purity suitable for crystallization trials upon request. Please contact us for more information.

o     Evaluation of the protein solubility (mg/mL).
 

o    Generally, 10 mg or more samples are preferred for initial screening. Crystallization experiments are designed according to the amount of samples available. We could also work with less samples using tailor-made crystallization screen based on the solubility of the samples. Please contact us for more information.

o    A small amount of sample is needed to determine its concentration. In addition, we can test its purity, stability and assemble state using SDS-PAGE, dynamic light scattering and mass spectrometry upon request.

Initial crystallization conditions are further optimized by exploring the following factors to obtain single crystals of diffraction quality:
               o         pH and buffer
               o         Protein concentration
               o         Precipitant 
               o         Detergent and additive
               o         Ligand
               o         Rate of diffusion
               o         Temperature
               o         Size and shape of the drops
               o         Pressure (e.g. micro-gravity)

For co-crystallization project, co-crystals of protein-ligand complex are formed by either co-crystallization methods or by soaking the preformed protein crystals with the ligand.

After obtaining single crystals, suitable cryoprotectant and cooling method are selected to obtain the highest diffraction resolution.

X-ray diffraction data are collected either using powerful, automated in-house Rigaku X-ray equipment or synchrotron radiation if necessary.

Depending on the phasing methods, various sets of data are collected:

    o        If the structure of a homologous protein is available in the RCSB Protein Data Bank (PDB;http://www.rcsb.org/pdb/home/home.do), one set of diffraction data is collected for structural determination by Molecular Replacement (MR).

    o        When a homologous model is not available, phase is determined by either Multiple Isomorphous Replacement (MIR) using heavy atoms or Multiple Wavelength Anomalous Dispersion (MAD) using selenomethionine derivatives. Depending on the phasing methods, more than more data sets are collected.

               o        For co-crystallization experiment of protein with known structure, one data set is collected for each ligand.
 

Our specialists and scientists have extensive experience in structure determination and refinement. After obtaining the correct initial phase, modeling and refinement are conducted in iterative cycles till R-factor converges to an appropriate low value with appreciable geometry of the atomic model. The quality of the final model will be validated by the validation programs, such as PROCHECK. Upon request, we can also help deposit the final structure into PDB, conduct detailed structural analysis and provide high-quality figures suitable for publication.