Background: The functions of proteins, the main elements of life activities, are fulfilled by their specific 3D structures. The structure determination of drug target proteins is necessary for the understanding of their biological functions, analysis of mechanism of action, and protein structure-based drug development. Sanyou has launched one-stop structure determination services covering from material preparation to structure determination of protein complexes and functional analysis.
Customized project design
The plan of structure determination will be designed based on client's needs and sample characteristics to provide customized services.
Analysis with two mainstream technologies
The success rate of protein complex structure determination is improved by the dual-technology platform of X-ray crystallography and cryo-EM.
Sample preparation and optimization with multi-system
The preparation of test sample is optimized in multiple aspects with QC criteria including various expression systems, purification regimens and activity tests.
High-resolution protein structure determination
Sanyou provides one-stop services from material preparation and sample optimization, to protein structure determination and functional analysis.
High quality and ultimate cost-performance
The preparation of samples for structure determination takes only 3–4 weeks. Extreme cost-performance and qualified delivery are guaranteed by the professional teams.
1. Sanyou's feature: structure determination assisted by super-trillion antibody libraries
With the super-trillion antibody libraries, Sanyou has unique advantages in antibody screening and discovery. Firstly, antibodies can effectively stabilize the structure and conformation of antigens as in a complex; in addition, the structure determination of the antigen-antibody complex is also necessary for the functional analysis of antigens. Therefore, with the assistance from Sanyou's super-trillion antibody libraries, we are able to provide numerous lead antibodies to stabilize the structure of antigen proteins; meanwhile, the analysis of epitopes where different kinds of antibodies (agonistic or antagonistic) bind to the antigen is helpful to understand the specific mechanisms of antibodies, thus providing new perspectives and insights for clinical drug R&D.
Fig. 1 structure determination assisted by super-trillion antibody libraries
2. Sanyou's feature: Structure-based drug screening
During the drug R&D, high-throughput screening is usually performed through drug reactivity to the active sites of the target. To be more specific, if the 3D structural information of a target protein is elucidated, its active site could be determined by point mutation or structure analysis. Subsequently, based on the molecular docking or molecular simulation of the candidate drug and the target protein, the intensity of the interactions is determined, and thus the possibility of drug binding is defined.Sanyou possesses the cutting-edge Alphafold2 structure prediction platform, and completes drug screening based on the simulation of molecular docking, so as to accelerate drug screening from the fundamental to help drug development.
Fig. 2 Structure-based drug screening
3. Sanyou's feature: In silico analysis of drug developability and immunogenicity
The differences in the structural characteristics of different antibodies can affect the drug developability and immunogenicity of the candidate antibodies, which is critical for successful drug development. In silico analysis of structure may help to understand the drug developability and immunogenicity of antibodies, and provide solid theoretical support for antigen design. Sanyou possesses an integrated QA system with full consideration of the drug developability and immunogenicity, which facilitates successful R&D of new drugs.
Table 1 In silico analysis of drug developability and immunogenicity
4. Sanyou's feature——AI-assisted protein design
Sanyou has a self-developed prediction platform based on Alphafold2 neural network, which can quickly predict the three-dimensional structure of an unknown protein or antigen-antibody complex structure, and provide solid theoretical support for antigen design with full consideration of the secondary structure and domain composition of proteins, as well as predict potential problems such as protein post-translational modifications.
Fig. 3 Alphafold2-assisted protein design
5. Material preparation with multi-systems
5.1 Diverse protein expression systems
The platform possesses E. coli prokaryotic expression system, mammalian cell expression system represented by CHO and HEK293 cells, and insect expression system represented by Sf9. The diverse expression systems can ensure the optimized expression of proteins. Simultaneously, the automated and integrated operation modes of the platform ensure efficient and rapid executions of the project, to improve the delivery efficiency.
Fig. 4 Diverse protein expression systems
5.2 Multi-path protein purification methods
The platform adopts methods including affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, and density gradient centrifugation. The homogeneity and purity of samples are improved by a combination of purification methods that comprehend the differences in surface charge or molecular weight of proteins. The platform provides customized purification protocols based on different expression systems and yields.
X-ray analysis of protein crystals shows a high resolution, which is suitable for proteins with molecular weights below 100 kDa, but requires a high concentration of protein samples and requires the growth of protein crystals with diffraction qualities. For the data collection of crystals, recoil method of monochromatic X-ray is commonly used, where the crystal rotates or swings around a small angle of axis in a certain vertical X-ray incidence direction, with the diffraction point recorded on the surface detector. Considering the existence of diffraction blind zone, it is necessary to optimize the swing angle. Thus, the collected data of crystals is essentially diffraction data.
Fig. 6 data collection X-ray crystallography diffraction
6.2 Cryo-EM transmission
The size of protein complex analyzed by Cryo-EM is generally more than 100 kDa. Cryo-EM is suitable for analyzing the structures of multiple protein complexes or of the virus under different conformations, but also requires high homogeneity and purity of the sample. For the data collection of frozen samples, the high-voltage electron beam penetrates the sample in the form of parallel light by electromagnetic field, and the scattering signal is recorded in the form of photographs using the detector and lens system. For the structure determination of frozen samples, the basic principle is the central section theorem, that is, the Fourier transform of an object projected in a certain direction is equivalent to cross section perpendicular to the projection direction of the object after the three-dimensional Fourier transform.
Fig. 7 3D reconstruction principle of Cryo-EM
1. Protein complex structure determination
Background: Structure determines function. The structure determination of protein complex is necessary to understand their physiological functions and dynamic conformational changes, especially structure-based functional analysis and drug design, which are hot spots in drug research and development.
Methods: X-ray crystallography diffraction or cryo-EM transmission was used for structure analysis, to further obtain the electron density map for establishing the atomic model. Then the obtained pdb structure files were used for subsequent functional analysis.
Results: As shown in Figure 1, the yellow ribbon diagram and purple ribbon diagram represent different proteins, respectively. The structure determination can define the domain composition and key secondary structure of the two proteins, of which the interaction and key combined amino acid sites can be further determined.
Fig. 8 Structure determination
2. Epitope analysis with antigen-antibody complex
Background: Antigen-antibody binding depends on specific epitope and spatial structure. Identifying structural information between the antigen and antibody, especially epitope analysis, is important to determine the function of the antibody.
Methods: A structure analysis software, Chimera, was used to analyze the interaction interface of the obtained pdb structure files. A spatial distance in the range of 5 Å was selected, the amino acid residues in this range are revealed to have interactions.
Results: As shown in the right panel, the red region shows the amino acid residues involved in the interaction on the antigen, and the yellow region shows the amino acid residues involved in the interaction on the antibody. The interaction mode (hydrogen bond, salt bond, and van der Waals force) between the antibody and antigen can be further determined by the software.
Fig. 9 Epitope analysis
3. Surface charge analysis of protein complexes
Background: Protein surface charge distribution can affect its stability and binding ability to other proteins, as well as the success of its drug development. Analyzing the charge distribution of unknown proteins is also necessary to understand their biological functions.
Methods: Chimera was used to analyze the surface charge distribution of the obtained pdb structural files.
Results: As shown in the right panel, the red region shows negatively charged amino acid residues, the blue region shows positively charged amino acid residues, and the white region shows amino acid residues in an electroneutral environment.
Fig. 10 Surface charge analysis
4. Hydrophobicity analysis of protein complexes
Background: The distribution of hydrophobicity on the surface of protein can affect the normal expression and purification of protein, and also change the interactions with other proteins.
Methods: Chimera was used to analyze the surface hydrophobicity of the obtained pdb structural files.
Results: As shown in the right panel, the blue region shows hydrophilic amino acid residues, the orange region shows highly hydrophobic amino acid residues, and the white region shows amino acid residues in a ralatively neutral environment.