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X-ray crystallography is currently the most favored method for structural determination of proteins and other macromolecules. The requisite for a successful X-ray crystallographic application is to obtain single crystals of the target protein. Data is then collected by diffracting X-ray from the single crystal that has an ordered pattern of atomic orientation. The assembly of atoms and molecules in the crystal can be deduced from the measurement of X-ray scattering.
At present, more than 120,000 protein structures resolved by X-ray crystallography experiments have been deposited in protein databank, accounting for nearly 90% of the resolved proteins, suggesting the predominant popularity of X-ray crystallography in structural determination.
Creative Biostructure provides X-ray crystallography services in our state-of-the-art facilities and has developed an X-ray crystallography pipeline that covers all technical stages from gene synthesis to structure determination. Our experienced scientists work closely with the clients to ensure rapid turnaround and reliable results.
|1. Construct design and expression optimization|
Plasmid construction: full-length, site-mutated, truncated, appropriate tag
Protein expression system: bacterial system, yeast system, insect cell/baculovirus system, mammalian cell system, plant system, cell-free protein production
|2. Crystallization-grade protein purification|
Proteins are purified using AKTA (GE Healthcare) with multiple chromatographic methods, such as ion exchange, affinity, size exclusion.
Protein purity is confirmed by SDS-PAGE, Western Blots, and/or Mass Spectrometry.
|3. Protein initial crystallization screening |
Initial crystallization screening by using the high-throughput crystallization analyzer and a variety of crystallization methods such as vapor diffusion crystallization, seeding, and co-crystallization, hundreds of non-redundant crystallization conditions can be screened.
|4. Optimization of crystallization conditions|
Parameters to be optimized include precipitant type, precipitant concentration, pH & buffer, protein concentration, additives, detergents, ligands, drop volume & drop ratio, temperature, pressure, etc.
|5. X-ray screening and dataset collection|
X-ray diffraction data is collected using powerful in-house Rigaku X-ray spectrometer or synchrotron radiation. Multiple datasets can be collected according to different methods, such as Molecular Replacement (MR), Multiple Isomorphous Replacement (MIR), and Multiple Wavelength Anomalous Dispersion (MAD).
|6. Data analysis and structure determination|
Electron density map
Model building and refinement
Model quality verification
Our services come with well-defined methodologies and instruments yet offer a great deal of flexibility upon customer's requirements. We can prepare crystallization-grade proteins from scratch or obtain them from customers. Besides, Creative Biostructure has multiple ready-made high-purity proteins and crystal structures developed internally that can be used for co-crystallization and compound soaking.
Creative Biostructure can provide various crystallization strategies, particularly for membrane protein crystallization. The detailed services are summarized as follows.
|Featured Crystallization Services||Feature|
|Co-crystallization||Co-crystallization retains the unique crystalline structures with their multiple components (e.g., proteins, DNA/RNA, chemical compounds, metal ions).|
|Bicelle-Protein Crystallization||Providing a more bilayer-like environment for membrane proteins than in detergent micelles, enabling the use of standard crystallization screening methodology for membrane proteins.|
|Lipidic Cubic Phase (LCP) Crystallization||LCP has a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment.|
|Controlled In Meso Phase (CIMP) Crystallization||CIMP stabilizes membrane proteins in meso phase and allows for direct monitoring of phase transformation and crystallization events.|
|Trace Fluorescent Labeling Crystallization||Great for the detection and identification of crystals by covalently labeling a fluorescent probe on the protein.|
|Crystallization Chaperone Strategies||Co-crystallization of challenging membrane protein targets with soluble protein chaperones.|
|Crystallization with Mutant Library Approaches||Improvement of protein solubility and crystallization assisted by mutant library construction and screening.|
Please feel free to contact us to discuss your project!