Cryo-Electron Microscope Technology: Referred to as Cryo-EM, applicated with cryo-ﬁxation technology, use Transmission Electron Microscope (TEM) to auto-image samples at low temperature and reconstruct the structure of biological macromolecules. Protein sample is cryo-ﬁxed in an ultra-thin vitriﬁed ice layer and placed under Cryo Transmission Electron Microscope, permitting the low dose beam to transmit through protein and nearby ice, then acquire the images. Firstly, the scattered signal is recorded by the detector and lens system, and then the signal is processed by computer. Finally, the D structure of the sample is obtained by using the D reconstruction technology.
- Provide a “one-stop service” from gene to protein structure. The deliverable data conforms to the PDB ﬁle submission criteria.
- Talos 200 kV Cryo Electron Microscope.
- Advanced Titan Krios 300 kV Cryo Electron Microscope analytical service is available.
- Basic information analysis services: crucial amino acid residue analysis, homology alignment, mutation design, and subsequent functional test program.
Within 3-6 months from image acquisition to data analysis, excluding sample preparation.
The negative staining and vitrified sample preparation is project-dependent.
The protein sequence can be directly provided.
Do not freeze and thaw the sample repeatedly. Keep it at 4 ℃.
|negative staining||vitriﬁed sample|
|＞0.025 mg，transmembrane protein＞0.1 mg||＞0.1 mg|
|Buﬀer: 1 mL, without glycerol and other organic solvents, salt ion concentration is less than 300 mM|
Frequently Asked Questions
How to evaluate the result of negative staining?
An ideal negative staining image should have a uniform sample distribution, and the staining plaques shown with no discernible edges at a magniﬁcation of 10, 000 times. The white staining plaques turn from deep to shallow ranging from center to the edge. At a magniﬁcation of 30, 000~40,000 times, the sample is well contrasted with the background and soft biological structures can be ob- served. Negative staining is proved to be a failure if the plaques appear distinct from black and white, lack of intermediate transition tones from light to dark, or has granular clumps on bright meshes.”
What is the cause of ice crystal contamination during the preparation of vitriﬁed samples?
Ice crystal pollution is a common problem in the production of vitriﬁed samples. There are three main reasons for the formation of ice crystal pollution: a. Insuﬃcient cooling rate； b. The temperature of coolant is not close to the freezing point； c. Water in the air condense on the surface of the sample or small ice crystals at the edge of the container fall into liquid nitrogen.”
What are the advantages of Cryo Electron Microscopy technique?
The sample does not need crystallization and has no phase problems. The structure of protein with high molecular weight can be resolved.”
Is it necessary to test the protein sample before sending it?
Yes, the SDS-PAGE (polyacrylamide gel electrophoresis) result diagrams of protein samples, LC-MC (liquid-mass chromatography) result diagrams and gel ﬁltration chromatography diagrams are all required for project evaluation to conﬁrm sample quality.”
Negative Staining Results Display
The negative staining results showed that the sample size was uniform and did not stack. The sample was stained as white and the biological structure could be ob- served. Negative staining could detect the quality of samples and determine the preparation concentration of vitriﬁed protein samples.
Vitriﬁed Protein Sample Preparation.
The vitriﬁed sample was prepared by loading the protein sample onto the electron microscope grid with porous carbon ﬁlm and placing the grid in liquid ethane cooled with liquid nitrogen. Qualiﬁed vitriﬁed samples must be free of ice crystals and free from contamination.
Firstly, the rotation and translation errors are eliminated through image matching, and the image is classiﬁed according to the princi- ples of FCS (Fuzzy Compactness and Separation). In the end, the particle-like images can be classiﬁed and the signal-to-noise ratio can be improved, so as to realize the reconstruction of high-resolution D structure. By classifying D images, software can be used to reconstruct D images.
The 3D reconstruction of the model is to use the central section theorem to determine the orientation of each particle image (orienta- tion determination). After comparing the projected reference image with the experimental particle image, spatial bearing parameters are obtained and new 3D structure is constructed. Finally, the spatial orientation of the experimental image is modiﬁed to form an iter- ative process until the ﬁnal 3D model is obtained.