Creative BioStructure is specialized in carrying out knowledge-based design of protein expression constructs that lead to high-yield expression of target proteins with biological activity. Depending on the design, the gene of the target proteins can be either purchased or synthesized in house.

In addition to expressing protein of biological function at crystallization quality, we are capable of making selenomethionine derivatives for MAD phasing.

Thousands of crystallization experiments are performed 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 further optimized in house or transmitted to the customers.

For initial screening, the client will provide the following information and materials if the macromolecules to be crystallized 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     Estimation of the protein solubility.
 

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 sample using
      tailor-made crystallization screen based on the solubility of the samples. Please contact us for more
      information.

o    A small fraction of sample is needed for concentration determination and quality assessment by
    Creative BioStructure. In addition, we can test samples for their purity, stability and aggregation state
      using SDS-PAGE, dynamic light scattering and mass spectrometry upon request.

Initial crystallization conditions are further optimized by exploring the following parameters 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.
 

Equipped with the comprehensive structure determination and analysis software programs, our staff scientists have extensive experience in structure determination and refinement. After obtaining the correct initial phase, model building and refinement are carried out 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.

Clients will pay a small amount of start-up cost to initiate the project. The rest of the payment is success-based for each stage. Detailed reports are provided for each stage and for the whole project. Due to the nature of X-ray Crystallography, the success can not be guaranteed. Consequently, clients are not under any obligation to pay for the stages that are not successful.

Please feel free to contact us to discuss your project and how we can customize our service to meet your research needs.

We will, using every possible means, reduce your risk!

This service is strictly confidential; disclosure of the identity of the protein under study is not required. The intellectual property rights related with crystal structure and crystallization conditions will be fully under clients.