Cryo-electron microscopy (Cryo-EM) has revolutionized structural biology, enabling researchers to visualize the intricate 3D structures of biological macromolecules at near-atomic resolution. This powerful technique is invaluable for understanding biological mechanisms and accelerating drug discovery and vaccine development. However, obtaining high-quality, high-resolution Cryo-EM data relies heavily on meticulous sample preparation. Two crucial components in this process are cryo em grids and the technology used for rapid freezing, such as a Vitrobot. ShuimuBio, a leading Cryo-EM service provider, leverages state-of-the-art facilities, expert teams, and innovative solutions like their proprietary GraFuture™ cryo em grids to overcome common sample preparation challenges and achieve exceptional results.
Cryo-EM Single Particle Analysis (SPA), a widely used Cryo-EM technique, involves capturing thousands or millions of 2D images of purified biological macromolecules and using computational algorithms to reconstruct a high-resolution 3D structure. SPA is particularly effective for resolving the 3D structures of challenging targets, including proteins that are difficult to crystallize. It allows researchers to keep samples in a near-native state, capture various conformational states, and determine the structure of heterogeneous protein complexes. Applications of Cryo-EM SPA span a wide range, from proteins like membrane proteins (GPCRs, ion channels, transporters), enzymes, and ribosomes, to DNA and RNA structures, protein-nucleic acid complexes, and various virus particles. ShuimuBio offers a comprehensive “one-stop” SPA solution covering various sample types including antibody-antigen complexes, small molecules & targets, PROTAC, membrane proteins, VLPs, and peptides.
A critical step in the Cryo-EM workflow, particularly for SPA, is rapidly freezing the sample in a thin layer of vitreous (non-crystalline) ice on a cryo em grid. This vitrification process is essential to preserve the sample’s native structure and prevent ice crystal formation, which can damage the specimen and obscure structural details.
The Role of Plunge Freezing Technology
To achieve rapid vitrification, samples are typically applied to a grid and then quickly plunged into a cryogenic liquid, often liquid ethane or a mixture of ethane and propane, cooled by liquid nitrogen. This process is known as plunge freezing. While simple in concept, achieving a uniform, thin layer of vitreous ice suitable for high-resolution imaging manually can be challenging.
Automated plunge freezers, such as the Vitrobot cryo em system, were developed to standardize and improve the plunge freezing process. A Vitrobot cryo em offers precise control over several critical parameters, including temperature, humidity, blotting time, and blotting force. The device typically operates within a controlled environmental chamber to maintain consistent temperature and humidity, minimizing ice formation from the air (frost). The user applies a small volume of sample to a cryo em grid held within the Vitrobot cryo em. Excess liquid is removed by blotting with filter paper, and the grid is then automatically and rapidly plunged into the cryogen. This automation and control significantly improve the reproducibility and quality of the vitrified ice layer compared to manual methods.
The sources state that ShuimuBio’s 24-hour machine time service platform is equipped with auxiliary equipment including Vitrobot and plasma cleaner. This indicates that ShuimuBio utilizes automated plunge freezing technology, such as the Vitrobot cryo em system, as part of their advanced setup to facilitate sample preparation for data collection.
Cryo-EM Grids: The Foundation for Imaging
The cryo em grid serves as the support structure for the vitrified sample during imaging in the electron microscope. Traditional cryo em grids typically consist of a metal mesh (often copper, gold, or molybdenum) with a thin support film, such as holey carbon or Quantifoil. While effective for many samples, these traditional grids can present challenges for certain types of biological specimens.
The sources highlight several issues encountered with traditional support grids during sample preparation for Cryo-EM:
- Air-liquid interface absorption
- Severe preferred orientation, where particles align in a limited number of orientations, making 3D reconstruction difficult
- High sample concentration threshold, often requiring concentrations above 1µM
- Significant background noise
- Difficulty reconstructing structures of “small” macromolecules
These challenges can lead to poor particle distribution, insufficient particle numbers, or preferred orientation on the grid, all of which negatively impact the quality and resolution of the final 3D structure.
GraFuture™ Grids: An Innovative Solution for Cryo-EM Sample Prep
Recognizing the limitations of traditional cryo em grids, ShuimuBio has developed its own series of graphene-supported membrane grids called GraFuture™. GraFuture™ represents an innovative solution specifically designed to overcome the sample preparation bottlenecks encountered with conventional methods.
GraFuture™ grids are designed to address several critical issues:
- They offer a potential solution to the problem of sample preferred orientation.
- They are suitable for applications where proteins have small molecular weights or low concentrations.
- They help mitigate strong background noise.
- They minimize damage caused by the air-liquid interface.
The GraFuture™ series includes GraFuture™ GO (Graphene Oxide) and GraFuture™ RGO (Reduced Graphene Oxide) supported membrane grids. The development of GraFuture™ grids is an independent research achievement by ShuimuBio. Utilizing these advanced cryo em grids, alongside their AI algorithm, significantly enhances the efficiency and precision of structural analysis. The specific advantages of choosing GraFuture™ grids, as listed in the sources, include improving sample preparation results and contributing to higher quality data.
By offering GraFuture™ grids, ShuimuBio provides researchers with a powerful tool to prepare challenging samples more effectively for Cryo-EM analysis, thereby increasing the likelihood of obtaining high-resolution structures.
ShuimuBio’s Integrated Cryo-EM Platform
ShuimuBio, founded in 2017, is Asia’s first commercial platform providing Cryo-EM structural analysis services. Their core team consists of experienced life and computational scientists and industry experts. The company has built an advanced and reliable experimental operation and data analysis workflow, supported by deep scientific and technological accumulation in structural biology from Tsinghua University.
ShuimuBio’s platform is designed to provide a seamless, “one-stop” solution from gene sequence to high-precision 3D protein structure. This integrated approach includes:
- Protein Preparation and Analysis Services: Offering various expression systems (E. coli, mammalian cells, insect cells, cell-free systems), purification processes, quality control (SDS-PAGE, Western blot, mass spectrometry, thermal stability, solubility), and protein assays (SPR, BLI, ELISA). They have deep experience in membrane protein production and purification.
- Advanced Cryo-EM Facilities: Owning globally scaled commercial Cryo-EM platforms with multiple 300kV microscopes (8 in total across Beijing and Hangzhou). These facilities are equipped with high-performance detectors, energy filters, and phase plates to ensure maximum imaging quality. The platform is supported by professional engineers for daily operation and maintenance, ensuring optimal performance. Auxiliary equipment, including Vitrobot and plasma cleaners, is part of their setup.
- Expert Team: A team of Ph.D. level scientists specializing in structural biology, protein science, and computational biology from top institutions.
- Rich Experience: Over 400 Cryo-EM projects completed, analyzed over 150 protein structures, with best resolution reaching 1.8 Angstroms. Over 300 single particle projects successfully completed. Structures resolved down to 51kDa. Achieved a breakthrough resolution of 1.4 Angstroms. Experience covers diverse samples like ion channels, GPCRs, antigen-antibody complexes, and spliceosomes.
- AI-Driven Platform: Independently developed SMART software series utilizes AI to enhance the efficiency of Cryo-EM data analysis. Their NanoSMART AI system automatically recognizes nanoparticle features in images, optimizing recognition even from low-quality images.
This comprehensive ecosystem, combining state-of-the-art equipment, expert knowledge, proprietary technology like GraFuture™ cryo em grids, and efficient workflows, positions ShuimuBio to tackle even the most challenging structural biology problems.
Why Optimized Sample Preparation is Key to High Resolution
Achieving high-resolution structures, like the 1.4Å and 1.8Å resolutions mentioned by ShuimuBio, requires consistently high-quality data collection. The quality of the data is fundamentally dependent on the quality of the prepared sample on the cryo em grid. Poor sample preparation, whether due to suboptimal freezing (where a Vitrobot cryo em or similar device is crucial), particle aggregation, denaturation, preferred orientation, or insufficient particle concentration, directly limits the achievable resolution.
By addressing these challenges with advanced methods and materials, such as carefully controlled plunge freezing using systems like the Vitrobot and utilizing specialized cryo em grids like GraFuture™, ShuimuBio maximizes the probability of obtaining grids suitable for high-resolution imaging. Their rigorous quality control system, based on Cryo-EM analysis and characterization, ensures samples meet research requirements.
The ability to consistently produce high-quality Cryo-EM samples allows ShuimuBio to leverage their powerful 300kV microscopes and AI-driven analysis pipelines to deliver the detailed structural insights needed for crucial research applications in vaccine development (including quality control and understanding antibody-vaccine interactions), antibody drug development (resolving antibody-antigen complexes, studying mechanisms, optimization), and small molecule drug discovery (target structure analysis, mechanism studies, fragment-based discovery).
In summary, the journey to a high-resolution Cryo-EM structure is complex, with sample preparation on cryo em grids being a foundational and often challenging step. Technologies like the Vitrobot cryo em automate the crucial plunge freezing process, while innovative cryo em grids like ShuimuBio’s GraFuture™ are specifically designed to overcome limitations of traditional grids, improving sample quality for difficult specimens.
ShuimuBio provides an end-to-end solution, combining these critical sample preparation technologies with world-class Cryo-EM facilities, expert scientists, and advanced AI software to deliver accurate, high-resolution structural analysis. Whether you are dealing with challenging membrane proteins, small molecules, or need rapid turnaround for your Cryo-EM projects, optimizing sample preparation with the right tools and expertise is paramount.
To learn more about how ShuimuBio’s integrated services, advanced cryo em grids, and state-of-the-art equipment can accelerate your structural biology research and help you achieve high-resolution Cryo-EM results, please visit https://shuimubio.com/. ShuimuBio is ready to assist with your next project.
