Creative Biostructure

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UniCrys™ Protein Crystallization Services (from Gene to Structure)

(1) Plasmid Construction 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.

UniCrys? Protein Crystallization Service (from Gene to Structure)

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:

  • Basic physical and chemical properties of the target protein including molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY).
  • Purity of the protein by SDS PAGE and/or Mass Spectrometry. We recommend a purity of at least 90% to 95% based on a Coomassie-stained SDS-PAGE. Creative Biostructure can further purify the submitted sample to crystallization purity upon request.
  • Evaluation of the protein solubility (mg/mL).
  • Ingredients of the protein storage buffer.

Samples:

  • Generally, 10 mg or more is preferred for the initial screening. Crystallization experiments are designed according to the available sample amount. We could also work with less samples using tailor-made crystallization screen based on the solubility of the sample. Please contact us for more information.
  • A small amount of sample is needed to determine its concentration. In addition, we can test its purity, stability and assemble state by SDS-PAGE, dynamic light scattering and mass spectrometry upon request.

(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.

UniCrys? Protein Crystallization Service (from Gene to Structure)

(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:

  • 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 can be collected for structural determination by Molecular Replacement (MR).
  • If a homologous model is not available, the phase can be determined by either Multiple Isomorphous Replacement (MIR) using heavy atoms or Multiple Wavelength Anomalous Dispersion (MAD) using selenomethionine derivatives. More than one set of data can be collected according to the methods used.
  • For co-crystallization of a protein with a known structure, one data set can be collected for each ligand.

(6) Crystal Structure Determination and Refinement

High-quality diffraction data can be further processed for indexing 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.

UniCrys? Protein Crystallization Service (from Gene to Structure)

Service Costs

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 deemed not 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.