Patent Number US.11,994,525.B2 ｜ Inventor: Dr. Atsushi Shimada
We provide technical licensing for cryo-electron microscopy grid preparation technology that directly forms lipid bilayers in the pores of carbon support membranes on electron microscopy grids, enabling the retention and data acquisition of membrane proteins within the lipid bilayers.
We resolve the challenges of conventional cryo-EM SPA of membrane proteins through our proprietary protocol.
Our unique approach of directly forming a lipid bilayer in the pores of a carbon support membrane breaks through the limitations of existing technologies and streamlines data acquisition.
In cryo-LSEM, lipid bilayers are formed directly in the pores of carbon support membranes on electron microscopy grids. Because lipid bilayers are thin, with a thickness of approximately 5 nm, this approach allows for reduced ice thickness compared to techniques using liposomes, enabling data acquisition at optimal ice thickness.
Membrane proteins are retained embedded within the lipid bilayer, allowing data acquisition in an environment closer to physiological conditions. This enables the capture of the three-dimensional structure of membrane proteins in a state more closely resembling their biological environment.
Additionally, our proprietary protocol concentrates membrane proteins on the grid, enabling data collection of sufficient number of particles even when the membrane proteins are present in trace amounts or at low concentrations in solution.
We have successfully achieved 3D reconstruction at 7.8 Å resolution even for unstable membrane protein complex samples at a low concentration of 0.03 mg/ml that were purified solely by affinity purification, which could not be structurally analyzed using conventional methods.
Our unique approach of directly forming lipid bilayers in the pores of carbon support films overcomes the limitations of existing technologies and streamlines data acquisition.
This technology targets membrane proteins. It is particularly suitable for samples with low yields that can only be processed up to affinity purification, samples that are difficult to concentrate, samples for which high-purity preparation is challenging, and unstable complexes formed by membrane proteins. This technology does not apply to soluble proteins.
When using 1 µl of sample at a concentration of 0.1 mg/ml, the calculation shows that membrane proteins will be densely embedded across the entire grid surface, so we recommend conducting experiments around this concentration. For lower concentrations, a larger volume of sample should be used, such as 5 µl. When 5 µl of 0.02 mg/ml sample is used, results equivalent to those obtained with 1 µl of 0.1 mg/ml sample can be expected. Based on our track record, we have successfully achieved 3D reconstruction with affinity-purified samples at approximately 0.03 mg/ml concentration, and we also have experience acquiring data at 0.02 mg/ml concentration. Although data acquisition may be possible at lower concentrations by using an appropriate sample volume, we have not attempted this. Since the optimal concentration for proper data acquisition varies depending on the sample, we recommend exploring concentration conditions within a certain range.
In LSEM, negatively stained samples tend to aggregate and appear clustered as the grid dries, since particles have a strong tendency to associate during the drying process. However, when grids are properly frozen for cryo-LSEM experiments, we have observed examples where dispersibility actually improved compared to conventional methods, suggesting that aggregate formation may be suppressed rather than promoted. Additionally, the aggregates formed when the grid dries in LSEM are not three-dimensional clusters but are, in principle, two-dimensional lateral associations of membrane proteins.
For membrane proteins with a molecular weight of approximately 500 kDa, we have not observed any recognizable preferred orientation problems. This is likely due to several factors: the lipid bilayer is not perfectly planar but exhibits fluctuations, the thickness of the lipid bilayer is not uniform, and membrane proteins are inserted at an angle into the lipid bilayer at a certain frequency. Additionally, by retaining membrane proteins in the lipid bilayer, we can expect an effect of keeping them away from the air-liquid interface, which may resolve the preferred orientation problems that typically occur in membrane protein samples using conventional methods. However, for large membrane protein complexes with a laterally extended morphology, preferred orientation problems arising from this technique may appear.
The types of condition optimization considered effective for cryo-LSEM are limited compared to conventional methods, such as membrane protein concentration and various parameters during grid freezing. We believe that through 1-5 grid preparation experiments, we can obtain the results of structure determination at the resolution achievable by cryo-LSEM for the sample in question, including success or failure. Therefore, we estimate that the number of licenses you will need to purchase by that time will be approximately 1-5.
Structural analysis of membrane proteins remains challenging even today. In fact, while over 6,000 membrane proteins are encoded in the human genome, as of the end of 2024, only approximately 250 human and mammalian membrane proteins have had their structures determined. Additionally, in structural analysis using cryo-electron microscopy single-particle analysis, the probability of successful structure determination is estimated at approximately 8% due to issues such as preferred orientation and aggregation. Furthermore, in the case of membrane proteins, high concentrations of approximately 1–5 mg/ml are often required compared to soluble proteins due to issues such as adsorption to carbon support films, and since membrane proteins are often difficult to concentrate, the success rate is considered to be even lower than that for soluble proteins. In contrast, cryo-LSEM is considered to enable sufficient data collection even at membrane protein concentrations of approximately 0.02 mg/ml, and we believe this method provides the best opportunity for structural analysis of low-concentration membrane protein samples. In fact, we have successfully concentrated a membrane protein sample at 0.03 mg concentration—which could not be analyzed by conventional methods—more than 90-fold on a grid and achieved 3D reconstruction at medium resolution. We have also successfully obtained a consistent density map at 7 Å resolution for E. coli AcrB using cryo-LSEM. Since consistent density maps have been obtained in both of the two cases attempted so far, we believe there is a high likelihood that cryo-LSEM simultaneously resolves issues such as aggregation and preferred orientation along with concentration problems. While high-resolution structural analysis has not yet been achieved, this is due to the limited number of case studies and experimental runs, and we expect that as cryo-LSEM is applied to a broader range of membrane proteins, the success rate for high-resolution structural analysis will become clear. Our company remains committed to sincerely supporting the success of our technology license purchasers and will continuously strive to improve success rates.
Generally, examination of samples using negative staining is typically performed first using conventional methods. We also believe that preliminary examination of sample properties using negative staining is effective in cryo-LSEM. However, even if aggregation is observed in examination using negative staining with LSEM, dispersibility may improve in cryo-LSEM. Therefore, there are cases where we proceed without confirming the properties of negatively stained samples using LSEM, and we have still achieved successful 3D reconstruction.
If you have prepared membrane protein solution, lipid solution, buffer, and other materials, the grid preparation experiment itself can typically be completed in 2 days with 3-4 hours of work per day using standard protocols.
In LSEM experiments using negative staining, we have confirmed the embedding into lipid bilayers for most of the more than 20 different membrane proteins tested so far.
No tagging or labeling of membrane protein samples is necessary.
Use is limited to the licensee's own research and business purposes. Redistribution, resale, or sublicensing to third parties is prohibited. Sharing or transferring serial numbers to third parties is also prohibited. Please refer to the Terms of Use for details.
We cannot process refunds or cancellations after purchase. Please review our Terms of Service and technical specifications thoroughly before making your purchase.
For inquiries before purchase, please use the form. You can consult with us about sample suitability and technical questions.
If you are interested in licensing for uses other than cryo-LSEM, such as imaging applications, or for specific drug discovery purposes or comprehensive collaborative research, please contact us using the contact form.
