Crystallization Screening


Structural Biology

  Address:Changshu High-tech Industrial Development district, Suzhou, Jiangsu Province 1001, Building 6, No.88 Xian Shi Road

      Protein crystallization is the process in which a highly pure and saturated protein sample packs itself in the form of a crystal consisting of millions of protein molecules arranged in a highly ordered fashion. It is usually the first step to determine the structure of the protein. The crystallization of protein molecules need demanding conditions. An excellent crystallization screening process is a prerequisite for obtaining good protein crystals, contributing to the research in protein crystallography and medicinal chemistry studies; facilitating the development of protein-based drugs, scaling up and optimizing production processes.

Service Content
Single crystal culture
Several methods can be used in crystal culture, such as solvent volatilization, cooling crystallization, vapour diffusion, liquid-liquid diffusion, sublimation and eutectic generation, and also, various combinations of parameters need to be adjusted for the crystal culture. Our team has a high level of expertise in crystal growth experiments and extensive experience in single crystal culture. We will develop the most suitable crystallization protocols according to the physico-chemical properties of the drug molecules.

Native recombinant protein crystal preparation

Suitable for samples with a known homologous protein structure.
If the amino acid sequence has a similar folding pattern to that of the homologous protein, the crystal structure of the target protein can be obtained by molecular replacement using the existing crystal structure of the homologous protein as a model, and then the crystal structure of the target protein can be resolved. These proteins are usually cultured using conventional crystal culture methods.
Crystal preparation by heavy-atom derivatization

Commonly used for samples lacking a known homologous protein structure.
The crystal diffraction data of natural proteins do not contain phase information. In the absence of a homologous protein structure model for molecular replacement, protein crystals containing heavy atoms can be cultured, and the phase information can be obtained by resolving the anomalous scattering signals of the heavy atoms in the crystals, and then the crystal structure of the target protein can be resolved.

Eutectic crystals are formed by complexes of proteins with other biomolecules (i.e. ligands such as nucleic acids, enzymes, small molecules and other protein molecules). The study of the pocket region by studying the eutectic structure of "protein-ligand" is important for understanding protein function, drug design and analysis of biomolecular interaction mechanisms. Eutectic structures are needed in other areas such as the development of small molecule drugs, antigen-antibody complexes, PROTAC, and the catalytic mechanism of enzymes. The preparation of eutectic is much more difficult than that of single protein crystals. In addition to conventional eutectic methods, ReadCrystal has a mature technology called MicroED, which breaks the bottleneck that the size of complex co-crystal is not enough for analysis, and only nanometer-sized crystals are needed to obtain the analysis data and obtain the complete structure.

Eutectic preparation
Co-crystallization method

Suitable for the study of “protein-ligand” complexes. The target protein is bound to the ligand to form a complex (e.g. antigen-antibody complex), which will be precipitated in the form of crystals after reaching a certain supersaturation state.
Conventional eutectic preparation methods include batch crystallization, liquid-liquid diffusion, vapor diffusion (suspension drop method, sitting drop method), etc.

Immersion method

A crystallization method base on the mechanism of interaction between a protein and its small molecule ligand.
Proteins are immersed in a small molecule ligand solution and the ligand diffuses into the binding site, resulting in eutectic crystals. The use of crystallography to study such eutectic structures provides access to the mechanisms of interactions of protein and small molecules and structural changes, and elucidates the principles of their molecular interactions.

Crystalline primary screening
In the initial screening stage, we usually use high throughput screening to obtain high purity protein crystals under different combinations of crystallization conditions, and subsequently observe the crystallization status by other techniques to determine the better crystallization conditions. This process requires adjustment of complex chemical and physical parameters including temperature, pH, dielectric constant of the solvent, salt in solution, and the presence and concentration of stabilizers.
ReadCrystal has dozens of crystallization kit combinations and nearly 2,000 crystallization screening conditions to achieve high throughput screening patterns in the primary screening stage. The application of highly efficient crystallization robots not only significantly reduces experimental time, but also reduces the amount of sample for a single crystallization primary screening condition to 0.1μL, reducing sample wastage and experimental cost.

Crystallization optimization
The size and quality of the crystallization sample directly affects the subsequent structural studies. Therefore, on the basis of the initial screening, we further optimize the conditions for protein crystallization by fine-tuning the crystallization parameters and adjusting the crystalline quality and solid form of the crystals to obtain optimal results.
The types of parameters that need to be adjusted in crystallization optimization are usually similar to the initial crystallization screening process. We optimize the sample by changing the precipitant, salt fraction, pH, dielectric constant of the solvent, temperature, etc. If the sample is difficult to form crystallization or you have special requirements for the crystallization process, we will apply special techniques such as the addition of detergents to enhance nucleation and crystal growth.

Protein crystal identification
Once crystals have been obtained, we should identify the protein crystals or salt crystals in order to reduce sample consumption and costs, improve efficiency, as well as optimize the crystallization conditions and thus improve product quality. Our crystallization laboratory is equipped with normal light microscope and UV microscope for the identification and screening of crystal samples.