The intricate world of biological macromolecules governs life itself, and unlocking their secrets requires understanding their three-dimensional structures. For decades, techniques like X-ray crystallography provided invaluable insights, but challenges remained, particularly for flexible, large, or difficult-to-crystallize complexes. This is where cryo-electron microscopy (cryo-EM) has revolutionized the field of structural biology and structure determination.

Cryo-EM allows researchers to visualize biological samples in a near-native, frozen-hydrated state. This is a significant advantage as it helps to preserve the natural state and can capture diverse conformational states that might be lost in crystallization. One of the most powerful applications of cryo-EM in structural biology is Single Particle Analysis (SPA). SPA integrates cryo-EM with computational algorithms to unveil high-resolution 3D structures of biological macromolecules like proteins and viruses. This process involves collecting numerous 2D images of purified particles, followed by computational processing and reconstruction to produce a detailed 3D structural model. The advantages of using cryo-EM SPA include preserving samples close to their native state, capturing diverse conformations, requiring minimal sample volume, and enabling the determination of heterologous protein complexes.

Navigating the complexities of cryo-EM structural biology and structure determination requires cutting-edge equipment, profound expertise, and efficient workflows. Shuimu BioSciences, founded in 2017 at Tsinghua University, stands as a pioneering commercial platform in Asia offering comprehensive cryo-EM structure determination services. The company is led by a core team of world-class experts and leverages the deep research and technical heritage in structural biology at Tsinghua University.

Shuimu BioSciences: A Leader in Cryo-EM Structure Determination

Shuimu BioSciences offers a "One-Stop SPA Solution" designed to streamline the entire structure determination process, saving time and costs. This solution encompasses the entire workflow from project consultation and feasibility evaluation to protein expression and purification, sample freezing and data collection, and finally, 3D reconstruction and model refinement, culminating in data delivery. They provide complimentary project feasibility assessments and maintain regular meetings to track progress.

Key to Shuimu's capabilities is its impressive infrastructure and team. They operate the world's largest commercial cryo-EM platform. This includes a significant number of 300 kV instruments dedicated to data acquisition, with two machines in Beijing and six in Hangzhou. Their Electron Microscopy Center is equipped with eight electron microscopes and full supporting apparatus, featuring high-performance detectors, energy filters, spherical aberration, and phase plates to ensure imaging quality. These instruments are meticulously maintained, boasting over 330 days of available machine time annually and an annual fault-free operation rate exceeding 97%.

Shuimu's commitment to high-resolution structural analysis is underpinned by their cutting-edge equipment and advanced computing platforms. Their team is composed of PhD-level experts from preeminent institutions, specializing in structural biology, protein science, and computational biology. This elite scientist team and extensive experience are crucial for successfully tackling complex structural challenges.

Overcoming Challenges in Cryo-EM Determination

Despite its power, cryo-EM can face challenges such as small protein molecular weight, low sample concentration, high background noise, disruption at the air-water interface, and preferential orientation. Shuimu addresses these issues through proprietary technologies like GraFuture™. GraFuture™ graphene support grids offer a potential solution to problems like preferred orientation and are suitable for applications involving small protein molecular weight, low concentration, strong background noise, and damage from the gas-liquid interface. They offer both Graphene Oxide (GO) and Reduced Graphene Oxide (RGO) versions of these grids.

Furthermore, Shuimu leverages its independently developed AI-driven platform, including the SMART software suite. This AI technology streamlines cryo-EM data analysis, reducing machine runtime and the required data volume, thereby improving the overall efficiency of the determination process. Their NanoSMART AI system is specifically designed for automatically identifying nanoparticle features in Cryo-Characterization applications.

Shuimu has accumulated extensive experience, completing over 400 cryo-EM projects since 2017. They have resolved more than 150 structures and successfully completed over 300 single particle projects. Their pursuit of resolution is uncompromising, achieving a best resolution of 1.8 Å and pushing the boundaries to achieve a groundbreaking resolution of 1.4 Å in some cases. They have successfully elucidated protein structures as small as 51 kDa, demonstrating their expertise in handling different molecular sizes.

Comprehensive Services Supporting Structural Determination

Effective cryo-EM determination often relies on high-quality sample preparation. Shuimu provides a complete protein expression and purification platform, including molecular cloning, expression using various systems (E. coli, mammalian cells, insect cells, cell-free expression), purification via methods like affinity, ion-exchange, gel filtration, and RP-HPLC chromatography, and characterization. This integrated approach minimizes variability from sample transport and standardizes the entire pipeline, addressing challenges posed by difficult-to-express proteins. Their expertise extends specifically to challenging targets like membrane proteins, including GPCRs, ion channels, and transport proteins.

Beyond cryo-EM SPA, Shuimu offers other complementary structural analysis methods, such as Micro-electron Diffraction (MicroED) for high-resolution structure determination from microcrystals and nanocrystals, particularly for small molecules, peptides, and protein crystals. Their proprietary eTasED software integrates MicroED into conventional cryo-EM systems, enhancing efficiency. They also offer One-Stop Crystallographic Analysis, covering protein expression, purification, crystallization, data collection, and analysis, which can reveal high-resolution structures of complexes like antigen-antibody interactions.

Initial assessment of sample quality and homogeneity is crucial for successful cryo-EM. Shuimu offers Negative Staining and 2D Negative Staining services to quickly obtain low-resolution two-dimensional projection images, assessing particle size, uniformity, morphology, and overall sample quality at a lower cost. They also provide Cryo Characterization services, leveraging their NanoSMART AI system for efficient and precise characterization of samples like AAVs, liposomes, and LNPs, providing detailed reports on size distribution, roundness, and integrity.

Further protein analysis services support the structural studies. Shuimu offers Protein Assays such as SPR (Surface Plasmon Resonance), BLI (Bio-Layer Interferometry), and ELISA (Enzyme-Linked Immunosorbent Assay). These techniques are vital for studying molecular interactions, including drug-protein binding, antigen-antibody interactions, and determining affinity and kinetics, complementing the structural information obtained from cryo-EM determination. Shuimu also maintains an Off-the-Shelf Protein List featuring important drug targets, including numerous membrane proteins, and offers customized protein target services.

Application Scenarios and Case Studies

The applications of cryo-EM structural biology are vast, impacting fundamental life sciences and drug development.

Analyzing Biomacromolecules: SPA can reveal high-resolution 3D structures of a wide range of biomacromolecules. This includes:

· Proteins: Membrane proteins like GPCRs (e.g., GPR75, GPR88, GPR35, GPR174), ion channels (e.g., TRPV4, TRPML1, Nav1.7), and transporters, as well as enzymes (e.g., CYP51, C21, PolQ, IMPDH2, GCH1, GFRP, p97, IDE, RNR) and ribosomes.

· DNA and RNA structures: Resolving double-helix structures, their interactions with other biomolecules, and analyzing viral RNA or ribosomal RNA.

· Protein–nucleic acid complexes: Such as transcription complexes (RNA polymerase bound to DNA) and viral capsid protein–RNA complexes.

· Viral particles: Including detailed structures of SARS-CoV-2 (like the S protein in complex with ACE2), Influenza virus surface spike proteins, African swine fever virus (ASFV), human herpesvirus 6B (HHV-6B), and rabies virus glycoprotein (VSV-GP).

Vaccine Field: Cryo-EM is indispensable in vaccine research and development. It helps in:

· Viral Structure Analysis: Resolving 3D structures at near-atomic resolution provides key insights for vaccine design by understanding viral entry mechanisms. Examples include determining the structure of the SARS-CoV-2 spike protein in complex with ACE2 and studying influenza virus surface proteins.

· Vaccine Quality Control: Examining morphology, particle size, integrity, and aggregation of vaccines across manufacturing stages helps optimize processes. Cryo-EM provides direct visualization and accurate measurement of aggregation levels.

· Antibody–Vaccine Interaction Studies: Clarifying how antibodies bind to vaccine antigens aids in optimizing immunogenicity. Studies resolving structures of vaccine-induced antibodies with targets like HIV-1 Env protein and influenza virus surface proteins provide structural bases for designing effective vaccines.

· Responding to Viral Mutations: Rapid structural analysis of new viral variants enables quick adjustment of vaccine designs, as demonstrated during the COVID-19 pandemic where cryo-EM was used to study SARS-CoV-2 variants.

Antibody Drugs: Cryo-EM plays a crucial role throughout antibody drug development. Its applications include:

· Antibody–Antigen Complex Structural Analysis: Revealing high-resolution structures to understand recognition mechanisms and binding sites, providing information for designing more effective antibody drugs.

· Mechanism of Action Studies: Studying how antibody drugs bind to targets and modulate signaling pathways. For instance, resolving the structure of an antibody targeting the Omicron variant of SARS-CoV-2 revealed the molecular basis of its activity.

· Optimization and Design: Analyzing existing drug structures to identify potential improvements, revealing dynamic processes during binding, and assisting in mapping conformational epitopes for antibody engineering.

· Structural Analysis of Membrane Proteins and Complex Targets: Resolving high-resolution structures of membrane protein targets like GPCRs, shedding light on ligand binding and receptor activation, which is crucial for developing antibodies against these targets.

· Accelerating Drug Development: The speed and clarity of cryo-EM accelerate the optimization of antibody designs and overall drug development processes.

Shuimu's platform has supported numerous research results published in top international journals, resolving atomic-level resolution structures covering ion channels, GPCRs, antigen-antibody complexes, spliceosomes, and other biological samples. Specific cases include the cryo-EM structure of the human GluN1-GluN2A subtype NMDA receptor bound with different small molecules and the structure of the H1R and rGq complex involved in histamine receptor activation. Their published work highlights successful determination of structures like human ACE2 to spike RBD from Omicron and Delta SARS-CoV-2, various GPCRs, ion channels, and antibody complexes.

Accessible and Professional Service

To support global academic and industry clients, Shuimu provides convenient and efficient 24-hour instrument time reservation services at their Beijing and Hangzhou centers. They offer expedited reservation channels and closed-loop management of the experimental process. Professional quality service includes daily platform maintenance by experienced technicians, ensuring equipment is always in optimal condition, and supporting online remote hole selection and real-time response to unexpected situations during data collection.

For clients interested in leveraging these advanced capabilities, Shuimu provides detailed sample submission requirements for various services, including protein solutions, small molecules, buffer solutions, cryo-samples, and crystals. Clear guidelines are provided regarding sample purity, concentration, volume, buffer composition, and shipping, all aimed at ensuring successful experiments.

In summary, cryo-EM technology has become an indispensable tool in modern structural biology, empowering researchers to achieve high-resolution structure determination of complex biomacromolecules in near-native states. Shuimu BioSciences offers a comprehensive suite of services, from sample preparation and characterization to advanced cryo-EM SPA, MicroED, and crystallography, supported by cutting-edge technology, an expert team, and efficient workflows, enabling rapid and accurate structural insights vital for life sciences and drug development.

To learn more about how Shuimu BioSciences can support your research in cryo-EM structural biology and structure determination, please visit https://shuimubio.com/.