X-ray Crystallography Services

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.

Advantages of X-ray Crystallography

  • To obtain a three-dimensional structure of protein/ protein-protein complex/ protein-small molecule complex with high atomic resolution; and to provide structural information is useful for understanding protein function and accelerates the process of rational drug design.
  • It is not limited by the molecular weight of the sample.
  • It helps reduce the technical barrier of obtaining single crystals of specific proteins, such as membrane protein, that are difficult to process by conventional methods.
  • The algorithm of protein structure analysis is well developed, and the established model has high confidence.

What We Do — Integrated Gene to Structure Services

Construct design and expression optimization1. 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
Crystallization-grade protein purification2. 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.
Protein crystallization and optimization3. 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.
X-ray screening and dataset collection4. 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.
Data analysis and Structure determination5. 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).
Data analysis and Structure determination6. Data analysis and structure determination

Phase 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 ServicesFeature
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.

Your Specific Sample Types for Crystallization

Please feel free to contact us to discuss your project!

References

  1. D'Arcy A, et al. Microseed matrix screening for optimization in protein crystallization: what have we learned?. Acta Crystallographica Section F: Structural Biology Communications. 2014, 70(9): 1117-1126.
  2. Hediger M R, et al. BioFET-SIM web interface: Implementation and two applications. PloS One. 2012, 7(10): e45379.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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