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Protein Crystallization

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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 a predominant popularity of X-Ray crystallography in structural determination.

The growth of structures from X-ray crystallography experiments deposited in PDB Fig.1. The growth of structures from X-ray crystallography experiments deposited in PDB

Creative Biostructure provides protein crystallization and 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.

Key features of this service?

  • 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 optimization

Step 1: Construct design and expression optimization

  • Plasmid construction: full-length, site-mutated, truncated, appropriate tag
  • Protein expression system: Escherichia coli system, yeast system, insect cell/baculovirus system, mammalian cell system, plant system, cell-free protein production
Crystallization-grade protein purification

Step 2: Crystallization-grade protein purification

  • Proteins are purified using AKTA (GE Healthcare) with multiple chromatographic methods including ion exchange, affinity, size exclusion.
  • Protein purity is confirmed by SDS-PAGE, western blots and/or Mass Spectrometry.
Protein crystallization and optimization

Step 3: Protein crystallization and optimization

  • Initial crystallization screening By using 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.
  • 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 and pressure.
X-ray screening and dataset collection

Step 4: X-ray screening and dataset collection

  • X-ray diffraction data is collected using powerful in-house Rigaku X-ray spectrometer or synchrotron radiation at our professional X-ray Crystallography Platform.
  • 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 determination

Step 5: Data analysis and Structure determination

  • Phase Determination
  • Electron density map
  • Model building and refinement
  • Model quality verification

Creative Biostructure not only conducts quality X-Ray crystallography services for customers all over the world, but also provides de novo preparation of protein crystals upon request. Our service plans come with well-defined methodologies and instruments, yet offer a great deal of flexibility. Please contact us for more information.

Creative Biostructure can provide a variety of crystallization strategies, particularly for membrane protein crystallization. The detailed services are summarized as follows.

Service 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. The fluorescent probe is concentrated along with the formation of crystals, producing fluorescent that is visually detectable under microscopic field.
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.


Crystal optimization solutions

Optimization Screening Optimization Screening
Batch Screening for Crystals Production Batch Screening for Crystals Production

Your specific sample types for crystallization

Optimization Screening Small Peptide
Batch Screening for Crystals Production Membrane Protein
Batch Screening for Crystals Production Antibody-Antigen Complex
Optimization Screening Viral Envelope Glycoprotein
Batch Screening for Crystals Production Discuss Details with Our Experts

Please feel free to contact us to discuss your project!

Ordering Process

Ordering Process

References

  1. D'arcy, Allan, et al. (2014) “Microseed matrix screening for optimization in protein crystallization: what have we learned?” Acta crystallographica. Section F, Structural biology communications 70 (Pt 9): 1117-1126.
  2. Martin R. Hediger, et al. (2012) “BioFET-SIM Web Interface: Implementation and Two Applications” PLOS one https://doi.org/10.1371/journal.pone.0045379.