Address：Changshu High-tech Industrial Development district, Suzhou, Jiangsu Province 1001, Building 6, No.88 Xian Shi Road
What is CryoEM-SPA?
With the development of structural biology, structure-based drug design has become the mainstream development paradigm for new drugs (i.e. screening potential binding molecules based on the structure of the target protein binding site). Cryo-electron microscopy single particle analysis (CryoEM-SPA), a microscopy technique that uses transmission electro microscopy to observe samples at low temperature, has received increasing attention and will gradually rival or surpass traditional crystal diffraction techniques to play a crucial role in biology, medicine and drug development.
Principle of CryoEM-SPA
CryoEM-SPA use electron scattering mechanisms to greatly reduce damage to molecular structures from high-energy electron beams through cryogenic freezing, and then convert the scattered signal into tens of thousands of single particle images and reconstruct the three-dimensional structure accordingly. In recent years, cryoelectron microscopy techniques have been enhanced to determine higher and higher resolutions, achieving near-atomic-level resolution of bio-macromolecule. CryoEM-SPA has become routine methods for resolving the structure of various protein molecules. CryoEM-SPA, together with X-ray crystal diffraction and NMR, form the basis of high-resolution structural biology.
Why choose CryoEM-SPA methods?
• Rapid frozen sample to preserve the native hydrated state of sample
• No crystallization, solve difficult membrane proteins in the industry
• Microgram-scale sample, high tolerance to protein purity
• Allows detection of multiple conformations of the same target protein
• Enables structural analysis of large protein complexes
Why choose ReadCrystal?
1、ReadCrystal provides one-stop customized services from gene synthesis, protein expression to cryo-electron microscopy structure analysis. We establish a team proficient in protein production and purification. We also have a team of electron microscopy experts.
2、ReadCrystal has its own 200 kV cryo-electron microscopy platform, equipped with Vitrobot cryo-robot. ReadCrystal also has sufficient commercial 300 kV cryo-electron microscopy time through in-depth cooperation with many institutions at home and abroad.
3、ReadCrystal provides timely response and communication programmed service at any time, which is convenient for customers to customize the plan, master the project progress and get the desired results.
1. Protein harvest: Customer can provide proteins by themselves or specify a protein so that we will express and purify the protein
2. Negative stain detection and assessment: a simple and rapid method for evaluating sample quality. Applying a heavy metal stain to the sample, with enhanced contrast, allows for a rapid visualization of the initial assessment of sample quality. This step is to assess the homogeneity of the sample (protein/complex state), dispersion, size and shape of the protein/complex, protein concentration (observation of the particle distribution).
3. Cryopreparation and evaluation:Before preparing a frozen sample, it is important to ensure that the sample has undergone a thorough biochemical purification process in order to have low structural heterogeneity and that the protein molecules are active. Cryopreparation is usually performed by passing a microsample (3-5μl) to Vitrobot, where a very thin layer of molecular solution is prepared and rapidly frozen by liquid ethane. After freezing, the water molecules in the sample do not form crystalline ice, but form an amorphous (glassy) ice layer. At this point the sample molecules will be better distributed in the ice layer. Before data collection, a diagnostic assessment of the frozen sample is still required, including protein concentration, distribution, stability, ice quality, thickness and homogeneity of the carrier network.
4. Data collection and processing:The highly stable 200kV and 300kV cryo-electron microscopes provide high throughput, automated data collection. Tens of thousands to millions of high-resolution single-particle images are acquired by the transmission electron microscope during this process, and the data is collated by specific algorithmic programs.
5. 3D reconstruction, model building: 3D reconstruction of macromolecules relies on averaging tens of thousands of particle images, a process in which the data is processed in multiple steps and requires the invocation of appropriate computational resources.