Understanding the intricate three-dimensional structures of proteins and other biomacromolecules is fundamental to advancing research in life sciences and drug development. This knowledge provides critical insights into their function, interactions, and mechanisms, which is vital for designing targeted therapeutics, vaccines, and understanding complex biological processes. Cryo-electron microscopy (cryo-EM) has emerged as a revolutionary technique for protein structure determination, offering significant advantages over traditional methods.

Founded in 2017 at Tsinghua University, Shuimu BioSciences stands as a leading commercial platform specializing in cryo-electron microscopy structure determination. Leveraging a core team of world-class experts and deep technical heritage in structural biology, Shuimu provides comprehensive "One-Stop" solutions, aiming to deliver high-resolution 3D structures from gene sequences.

What is Cryo-EM Single Particle Analysis (SPA)?

At the heart of many cryo-EM structure determination projects is Single Particle Analysis (SPA). This powerful approach integrates cryo-EM technology to reveal high-resolution 3D structures of biological macromolecules like proteins and viruses. The process involves collecting numerous 2D images of purified macromolecular particles, followed by computational algorithms for processing and reconstruction to produce a detailed 3D structural model.

The typical workflow for SPA includes Project Consultation & Discussions, Feasibility Evaluation, Strategy Definition, Contract & Payment, Protein Expression & Purification, Negative Staining (often used as an initial assessment step), Sample Freezing & Data Collection, 2D Particle Picking, 3D Reconstruction, Model Refinement, and finally, Data Delivery.

Why Choose Cryo-EM for Protein Structure Determination?

Cryo-EM offers several key advantages that make it ideal for resolving the structures of diverse biological samples:

· Preserves Samples Close to Their Native State: Cryo-EM allows samples to be studied in a near-native, hydrated state by rapidly freezing them in amorphous ice. This preservation is crucial for visualizing macromolecules in their functional conformations.

· Captures Diverse Conformations: Unlike methods that require crystallization, cryo-EM can capture multiple conformational states of a flexible protein or complex within a single sample. This is invaluable for understanding dynamic processes.

· Requires Minimal Sample Volume: Cryo-EM typically requires significantly less sample material compared to traditional crystallography.

· Determines Heterologous Protein Complexes: Cryo-EM is well-suited for determining the structures of large and complex assemblies, including protein complexes with multiple components.

· High Resolution Capabilities: Cryo-EM can achieve near-atomic resolution, providing detailed structural information. Shuimu's platform has achieved resolutions as fine as 1.4 Å.

· Overcoming Sample Challenges: Cryo-EM, especially when combined with specialized tools like Shuimu's proprietary GraFuture™ graphene support grids, can overcome challenges posed by samples with small molecular weight, low concentration, high background noise, air-water interface disruption, and preferential orientation. These issues can make structure determination difficult with other techniques.

Shuimu BioSciences: A Leader in Cryo-EM Structure Determination

Shuimu BioSciences is specifically purpose-built for high-resolution structural analysis using cryo-EM. Their platform offers cutting-edge equipment, an elite scientist team, extensive experience, and AI-driven solutions.

· Industry-Leading Equipment: Shuimu operates the world's largest commercial cryo-EM platform, featuring 300 kV instruments located in both Beijing and Hangzhou. As of the source information, this includes 2 instruments in Beijing and 6 in Hangzhou for 300 kV data acquisition, totaling 8 instruments mentioned in one section, although another section mentions 12 instruments in Beijing and 6 in Hangzhou for 300 kV data collection, totaling 18. These include models like G3i, G4, and Totem. The platform emphasizes daily maintenance and optimal performance.

· Experienced Scientist Team: The team is composed of PhD-level experts from preeminent institutions, specializing in structural biology, protein science, and computational biology.

· Extensive Project Experience: Since 2017, Shuimu has completed over 400 cryo-EM projects for clients, resolving more than 150 structures. They have successfully completed over 300 single particle projects. Structures have been resolved with exceptional resolution, with over 300 proteins resolved at <3.5 Å and a groundbreaking best resolution of 1.4 Å achieved. They have also successfully elucidated protein structures as small as 51 kDa. This extensive experience spans various sample types, including membrane proteins and antigen-antibody complexes.

· AI-Driven Platforms: Shuimu leverages proprietary AI algorithms and software to enhance efficiency and accuracy. Their independently developed SMART software suite streamlines cryo-EM data analysis, reducing machine runtime and required data volume. For cryo-characterization, the NanoSMART system automatically identifies nanoparticle features and provides detailed reports. For MicroED, the eTasED software integrates the technology into conventional cryo-EM systems, enhancing efficiency and accuracy.

· Specialized Tools: Shuimu offers GraFuture™ graphene support grids designed to overcome challenges like preferred orientation, suitable for samples with small molecular weight, low concentration, or high background noise. They offer GraFuture™ GO (Graphene oxide) and GraFuture™ RGO (Reduced graphene oxide) options.

Shuimu's commitment to quality is underpinned by a dedicated team of EM engineers overseeing daily operations and maintenance, ensuring data is collected under optimal performance. Their global customer support centre collects feedback to objectively evaluate project delivery.

Integrated Services for High-Quality Samples

Successful protein structure determination by cryo-EM relies heavily on the quality of the sample. To minimize variability from sample transport and standardize the entire pipeline, Shuimu has built a complete protein expression and purification platform alongside its cryo-EM services. This allows them to provide "One-Stop" solutions from gene sequences to high-resolution 3D structures.

Their protein preparation and analysis services include:

· Protein Expression Systems: Utilizing E. coli, mammalian cells, insect cells, and cell-free expression systems, allowing selection of the optimal system based on the target protein's characteristics (e.g., post-translational modifications, solubility, toxicity). The mammalian system is preferred for proteins requiring natural post-translational modifications, while E. coli is economical and fast. The insect cell system offers high expression efficiency and post-translational modifications, and the cell-free system offers rapid synthesis.

· Protein Purification Processes: Employing techniques like Affinity chromatography, Ion-exchange chromatography, Gel filtration, and Reverse-phase HPLC (RP-HPLC) to achieve high purity. They have extensive experience in producing and purifying difficult targets like membrane proteins (GPCRs, ion channels, transporters).

· Protein Quality Control: Rigorous QC is performed using methods such as SDS-PAGE, Western blot, mass spectrometry, and thermal stability and solubility testing. They specifically conduct protein quality control based on rigorous cryo-EM analysis and characterization to ensure samples meet research requirements.

· Protein Assays: Offering services like SPR, BLI, and ELISA for studying protein-protein or protein-molecule binding, crucial for understanding interactions and validating protein activity.

· Off-the-Shelf Protein List: Providing access to a list of important drug targets, including many membrane proteins, which can serve as starting materials for various studies.

This integrated approach ensures that researchers start with high-quality, well-characterized protein samples, significantly increasing the likelihood of successful cryo-EM structure determination.

Supporting Cryo-EM Services

Beyond SPA, Shuimu offers other cryo-EM related services that support structural studies:

· Negative Staining & 2D Negative Staining: This technique provides a rapid and lower-cost method to obtain low-resolution 2D images. It is used for initial assessment of sample particle size, uniformity, morphology, particle density, flexibility, and integrity before moving to cryo-EM. It is particularly useful for homogeneity assessments of particles like AAV, exosomes, membrane proteins, viruses, and soluble proteins.

· Cryo Characterization: This service uses ultra-low temperature to maintain the natural state of samples for high-resolution structural observation and analysis, especially useful for liposomes, LNPs, AAV, and other viral vectors. The NanoSMART AI system aids in efficiently and precisely characterizing these nanoparticles, including size distribution, roundness, and full/empty integrity.

· MicroED Solutions: Micro-electron diffraction is a cutting-edge technology for resolving high-resolution structures from microcrystals and nanocrystals, particularly suited for small molecule drugs, peptides, and protein crystals. Shuimu's eTasED software enhances this capability. They have achieved resolutions of 0.6~1.0 Å for MicroED projects.

· One-Stop Crystallographic Analysis: While cryo-EM is a primary focus, Shuimu also offers X-ray crystallography services for samples amenable to crystallization, covering protein expression, purification, crystallization, data collection, and analysis. This method is particularly effective for targets like antigen-antibody complexes, providing insights into their interactions.

Applications of Cryo-EM Protein Structure Determination

The ability to determine high-resolution protein structures using cryo-EM has wide-ranging applications.

· Analyzing Biomacromolecules: SPA can reveal the 3D structures of a variety of biomacromolecules, including many types of proteins (membrane proteins like GPCRs, ion channels, transporters; enzymes like CYP51, C21, PolQ; Ribosomes), DNA and RNA structures, protein-nucleic acid complexes (transcription complexes, viral capsid-RNA complexes), and viral particles (SARS-CoV-2, Influenza virus, ASFV, HHV-6B, VSV-GP).

· Vaccine Field: Cryo-EM is crucial in vaccine research and development (R&D). It helps resolve viral structures at near-atomic resolution, providing insights into viral entry mechanisms and guiding vaccine design. Examples include studies on SARS-CoV-2 spike protein/ACE2 interaction, strategies for attenuated live influenza vaccines, and how neutralizing antibodies block measles virus. Cryo-EM is also used for vaccine quality control, examining morphology, particle size, integrity, and aggregation. Furthermore, it helps clarify antibody-vaccine antigen binding, aiding immunogenicity optimization, as seen in HIV and influenza vaccine research. It also enables rapid structural analysis of new viral variants, supporting adjustments to vaccine designs, as demonstrated during the COVID-19 pandemic.

· Antibody Drugs: Cryo-EM plays a crucial role in antibody drug development. It reveals high-resolution structures of antibody-antigen interactions, aiding the understanding of recognition mechanisms and binding sites. It is used for mechanism of action studies, showing how antibodies bind targets and modulate pathways. Structural analysis helps optimize antibody designs, identify improvements, and map conformational epitopes for engineering. Cryo-EM can also resolve structures of membrane protein targets like GPCRs, providing vital information for developing antibodies against them. The speed and clarity of cryo-EM accelerate the overall drug development process. Examples include resolving the structure of a broadly neutralizing dual-epitope antibody against the Omicron variant.

Sample Submission Guidelines

To ensure successful structure determination, providing high-quality samples is essential. General requirements for protein solutions for SPA include concentration ≥ 2 mg/mL, volume ≥ 100 µL, and purity ≥ 90%. Specific buffer requirements, such as reducing glycerol, salt ions, and detergents, are noted. Minimizing repeated freeze-thaw cycles and using freshly prepared samples is recommended.

For cryo-sample delivery for instrument access, advance notification is required, and samples can be transported via liquid nitrogen tanks. For MicroED, stable crystals (powder or lump) are needed with sufficient quantity and purity. Specific sample requirements exist for other services like Negative Staining, Cryo-Characterization, SPR, BLI, and ELISA, detailed in the source material. Shuimu's scientists and sales colleagues are available for consultation on sample requirements.

Experience Highlights and Case Studies

Shuimu's expertise is demonstrated through numerous successful projects and publications. Their platform has supported research published in top international journals, resolving atomic-level resolution structures covering ion channels, GPCRs, antigen-antibody complexes, spliceosomes, and other biological samples. Case studies highlight the resolution of structures like the human GluN1-GluN2A subtype NMDA receptor bound with small molecules and the H1R and rGq complex. The sources also list numerous articles published by Shuimu's partners, showcasing the impact of their structural biology services on cutting-edge research.

In summary, cryo-EM, particularly cryo-EM SPA, is a powerful tool for determining the structures of proteins and other biomacromolecules, essential for life sciences and drug development. Its advantages in preserving native states, capturing conformations, and handling challenging samples make it indispensable. Shuimu BioSciences, with its advanced platform, expert team, integrated services, and proven track record, is equipped to handle complex protein structure determination projects using cryo-EM.

To learn more about their protein structure determination services, including cryo em and comprehensive protein preparation, purification, and analysis solutions, please visit https://shuimubio.com/.