(1) PlasmidConstruction and Protein Expression
Creative Biostructure is specialized in plasmid construction of full-length, site-mutated, truncated and tagged proteins. The constructed gene is cloned into a suitable vector and expressed in an appropriate cell line.
(2) Purification of Crystallization-grade Proteins
Proteins are purified using Akta (GE Healthcare) with multiple chromatographic methods including ion exchange, affinity, size exclusion, etc. Resins with specific affinity to the tags are used in the case of tagged proteins. Protein purity is checked via SDS-PAGE and western blots.
(3) Initial Crystallization Screening
Hundreds of non-redundant crystallization conditions will be screened by taking advantage of the high-throughput crystallization facility alongside multiple crystallization approaches, such as vapor diffusion crystallization, seeding and co-crystallization.
Creative Biostructure can de novo prepare the protein samples for crystallization with our UniCrys™ Gene-to-Structure Platform or obtain them from customers. In the latter case, the following information and material specifications are necessary for more efficient crystallization:
(4) Optimization of Crystallization Conditions
It is possible that crystals qualified for X-ray diffraction could be obtained just by the initial screening, while most of the time, an optimization process is necessary to get high-quality crystals. After the initial crystal screening, several working crystallization conditions will be further optimized to get bulk crystals: firstly, important experimental variables will be tested, such as pH, protein concentration, precipitants, detergents, additives, ligands, temperature, etc.; secondly, different approaches for crystal growth are also to be evaluated, e.g., seeding, cross-seeding and crystal re-growth techniques.
Creative Biostructure has the most advanced UniCrys™ in-house X-ray facilities, making it convenient to screen different crystallization strategies for diffraction studies. Suitable cryo-protectants and cooling methods are also used to obtain the best diffraction data.
(5) X-ray Data Collection
X-ray diffraction data is collected either using the powerful, automated in-house Rigaku X-ray equipment or synchrotron radiation if necessary. The manner of data collection can determine the data quality, and thus the success rate of subsequent steps in structure determination as well as the model quality. Our specialists on data collection are able to improve the diffraction quality by using cryo-protectant, anneal and dehydrant.
Depending on the phasing methods, various sets of data can be collected:
(6) Crystal Structure Determination and Refinement
High-quality diffraction data can be further processed forindexing and integration to data scale using available software. Since the X-ray detector can only record intensities but not phases of the electromagnetic waves, the phase problem is the trickiest part in determining a crystal structure. Different phasing techniques, which can provide a set of initial, approximate phases derived from additional experimental data, are used to find out the initial phases. They are:
After obtaining the initial phases, an electron density map can be computed, and the process of model building and refinement will begin. Model building and refinement are conducted in iterative cycles till the R-factor converges to an appropriate low value with an appreciable geometry of the atomic model. By this, we will provide a high-quality model that nicely fits the electron density map. The quality of the final model will be validated by programs like PROCHECK. Upon request, we can also help deposit the final structure into Protein Data Bank (PDB), conduct detailed structural analysis and provide high-quality figures for publication.
A small down payment is needed to get the project started, and additional ones could be made based on the progress of the project, for which progress reports will be prepared for the reading of the customers. Protein crystals or structures including X-ray data are delivered as the final products of the whole project. Due to the unpredictable nature of protein crystallization study, the project may be delayed or the final results deemednot suitable for publication. Should that ever happen, customers will not be charged for the stages that are not successful.
Please feel free to contact us for a discussion on your project. We will do our best to to serve your interests.
We will, using every possible means, save your time and money!
We keep all your information confidential, and claim no ownership on any experimental data and materials, once the project is accomplished.