GraFuture vs. Cryo-EM: Pros & Use Cases

Cryo-electron microscopy (cryo-EM), particularly Single Particle Analysis (SPA), has revolutionized structural biology by enabling the determination of high-resolution 3D structures of biological macromolecules. This powerful technique involves collecting numerous 2D images of purified macromolecular particles and using computational methods for processing and reconstruction to produce a detailed 3D structural model.

The advantages of Cryo-EM SPA are significant: it preserves samples close to their native state, captures diverse conformations, requires minimal sample volume, and can determine the structure of heterologous protein complexes. This makes cryo-EM vital for life sciences and drug development, offering high-resolution capabilities for investigating both the structure and functional roles of biological macromolecules, from single proteins to complex viruses.

Cryo-EM SPA can reveal high-resolution 3D structures of various biomacromolecules, including proteins like membrane proteins (GPCRs, ion channels, transporters), enzymes, and ribosomes. It is also applicable to DNA and RNA structures (resolving double helix, viral RNA, ribosomal RNA), protein-nucleic acid complexes (transcription complexes, viral capsid protein-RNA complexes), and viral particles (SARS-CoV-2, Influenza virus, African swine fever virus, human herpesvirus 6B, rabies virus glycoprotein). The applications extend into the vaccine field for viral structure analysis, vaccine quality control (examining morphology, particle size, integrity, aggregation), antibody-vaccine interaction studies, and responding to viral mutations. In antibody drug development, cryo-EM plays a crucial role in antibody-antigen complex structural analysis, mechanism of action studies, optimization and design of antibody drugs, structural analysis of membrane proteins and complex targets, and accelerating the drug development process.

Challenges in Conventional Cryo-EM Sample Preparation

While cryo-EM is powerful, traditional support grids used for sample preparation can present several challenges that act as bottlenecks. These issues can impede successful structure determination, especially for certain types of samples. The difficulties encountered with conventional methods include:

• Gas-liquid interface absorption: This can damage or denature delicate biological samples.
• Severe preferred orientation: Particles may orient themselves predominantly in one or a few directions on the grid, making it difficult to obtain a complete set of views needed for 3D reconstruction.
• High sample concentration threshold (>1μM): Many proteins are challenging to produce at such high concentrations or require low concentrations to maintain stability and activity.
• Significant background noise: Noise can obscure the signal from the particles, making data analysis more difficult.
• Difficulty in reconstructing "small" macromolecular structures: Samples with small molecular weights are harder to visualize and reconstruct at high resolution due to weaker signals.

These challenges can limit the types of samples amenable to high-resolution cryo-EM analysis and affect the quality and efficiency of the process.

For researchers seeking to overcome these hurdles and obtain high-quality structural data, explore Shuimu BioSciences's advanced solutions at https://shuimubio.com/.

Introducing GraFuture™: An Advanced Technique

To address the limitations posed by traditional sample preparation methods, Shuimu BioSciences has developed a series of graphene support grids known as GraFuture™. GraFuture™ provides a potential solution to some of the persistent challenges in cryo-EM sample preparation, particularly the preferred orientation problem.

The GraFuture™ line includes:

• GraFuture™ GO: Graphene oxide support grids.
• GraFuture™ RGO: Reduced graphene oxide support grids.

By using GraFuture™ support grids, researchers can benefit from their ability to mitigate issues such as small protein molecular weight, low concentration, strong background noise, damage from the air-water interface, and preferential orientation. These advantages offered by choosing GraFuture™ support grids directly correspond to overcoming the previously mentioned bottlenecks in conventional sample preparation.

How GraFuture™ Enhances Cryo-EM

The core idea behind GraFuture™ is to provide a support film that interacts differently with the sample compared to traditional carbon films, thereby influencing particle distribution and orientation. By providing a potential solution to the preferred orientation problem and being suitable for challenging conditions like low concentration and small molecular weight, GraFuture™ significantly enhances the capabilities of cryo-EM.

This innovation is part of Shuimu BioSciences's broader commitment to leveraging proprietary technology to improve cryo-EM efficiency and accuracy. Along with proprietary AI algorithms, graphene-based grids like GraFuture™ are listed among Shuimu's core strengths for structure determination.

Shuimu BioSciences: A Leader in Cryo-EM and Structural Biology

Shuimu BioSciences, founded in 2017 at Tsinghua University, is positioned as the first commercial platform in Asia offering cryo-EM structure determination. Led by experts in life sciences, computational science, IT, and pharmaceuticals, the company's EM Centre boasts experienced EM scientists and leverages the structural biology heritage of Tsinghua University to establish efficient, advanced workflows. Dedicated engineers ensure optimal performance of the cryo-EM facilities.

Shuimu offers a "One-Stop" approach, aiming to provide solutions covering everything from gene sequences to high-resolution 3D structures. This is supported by their expansion of the cryo-EM platform and the establishment of a complete protein expression and purification platform.

Core Strengths of Shuimu BioSciences include:

• Extensive Instrument Platform: Owning advanced biomolecular imaging equipment, Shuimu has a large commercial cryo-EM platform. The source mentions 2 × 300 kV instruments in Beijing and 6 × 300 kV instruments in Hangzhou as core strengths. Another section notes a 300 kV cryo-EM center equipped with eight electron microscopes and supporting apparatus, with 12 instruments in Beijing and 6 in Hangzhou for 300 kV data acquisition. While the total number cited varies (8 vs 18), the platform is extensive and globally accessible. Instruments mentioned include G3i, G4, and Totem for 300 kV data acquisition. These facilities provide 24-hour instrument access.
• Proven Experience: The platform has completed over 400 cryo-EM projects and resolved more than 150 structures.
• High Resolution Capabilities: Achieved a best resolution of 1.8 Å and successfully elucidated protein structures as small as 51 kDa. One case sharing highlights a groundbreaking resolution of 1.4Å. Structures resolved include ion channels, GPCRs, antigen-antibody complexes, and spliceosomes.
• Proprietary Technology: Utilizes proprietary AI algorithms (SMART software suite) and graphene-based grids (GraFuture™) to significantly enhance efficiency and accuracy. The AI-driven SMART software streamlines data analysis and reduces machine runtime. The eTasED software integrates micro-electron diffraction (MicroED) into conventional cryo-EM systems, enhancing efficiency and accuracy.
• Expert Team: Composed of PhD-level experts from preeminent institutions specializing in structural biology, protein science, and computational biology.

Shuimu's ability to achieve high resolutions and handle diverse samples, including challenging targets like membrane proteins and small macromolecules, is supported by both cutting-edge equipment and advanced techniques like GraFuture™ and their AI platform.

Learn more about Shuimu BioSciences's advanced cryo-EM services and how they utilize techniques like GraFuture™ to push the boundaries of structural biology by visiting https://shuimubio.com/.

Integrating GraFuture™ into the SPA Workflow

The standard Single Particle Analysis (SPA) workflow involves several steps: Project Consultation & Discussions, Feasibility Evaluation, Strategy Definition, Contract & Payment, Protein Expression & Purification, Negative Staining (often for initial assessment), Sample Freezing & Data Collection, 2D Particle Picking, 3D Reconstruction, Model Refinement, and Data Delivery.

Protein preparation and analysis services are crucial upstream steps, including expression systems (E. coli, mammalian, insect, cell-free), purification processes (affinity, ion-exchange, gel filtration, RP-HPLC), antibody discovery, protein quality control (SDS-PAGE, Western blot, mass spectrometry), and protein assays (SPR, BLI, ELISA). Shuimu offers extensive experience in membrane protein production and purification.

GraFuture™ grids would be utilized during the Sample Freezing & Data Collection phase, specifically during the grid preparation step where the sample is applied to the grid and vitrified. Their design aims to optimize particle distribution and orientation before data acquisition begins. This improved sample preparation step directly impacts the quality of the collected data and the subsequent success of 2D particle picking and 3D reconstruction.

Shuimu's platform ensures daily maintenance of equipment to guarantee efficiency and quality of data collection. They support online remote hole selection and scientist experience-based hole selection. Sample submission requirements for cryo-samples involve delivering them in advance, potentially using liquid nitrogen tanks, and notifying the operations manager.

Sample Considerations for Cryo-EM and GraFuture™

While specific sample requirements solely for GraFuture™ are not separately detailed, the source provides general requirements for protein solution samples for cryo-EM: concentration ≥ 2mg/mL, volume ≥ 100ul, and purity ≥ 90%. It is recommended to reduce glycosylation or phosphorylation modifications, minimize freeze-thaw cycles, and use freshly prepared samples. Buffer solutions should have reduced organic solvents like glycerol and salt ion concentration ≤ 300mM.

However, GraFuture™ is specifically highlighted as suitable for applications with small protein molecular weight, low concentration, strong background noise, and gas-liquid interface damage. This suggests that samples that might not meet the standard concentration requirement of 2mg/mL might be successfully prepared using GraFuture™ grids, leveraging their ability to handle low concentration conditions.

For optimal results when using advanced techniques like those offered by Shuimu BioSciences, including the potential benefits of GraFuture™ grids for challenging samples, understanding and meeting sample submission requirements is key.

To discuss the specific requirements for your challenging sample and explore how GraFuture™ can assist, contact the experts at Shuimu BioSciences via https://shuimubio.com/.

Beyond SPA: Other Complementary Techniques

Shuimu BioSciences also offers other structural analysis techniques that complement cryo-EM SPA, such as MicroED (Micro-electron diffraction) and X-ray Crystallography.

MicroED is a cutting-edge technology for resolving high-resolution structures from microcrystals and nanocrystals, particularly useful for small molecule drugs, peptides, and protein crystals. Shuimu has achieved resolutions of 0.6~1.0Å with MicroED projects and successfully resolved structures of small molecules, peptides (e.g., FUS LC, Acetaminophen), and proteins (e.g., Proteinase K). Their proprietary eTasED software enhances MicroED technology integration.

One-Stop Crystallography services cover the entire workflow from protein expression and purification to crystallization, data collection, and structure analysis using X-ray crystallography. This technique is valuable for resolving structures of antigen-antibody complexes, small molecule drugs, and peptides, particularly useful for understanding antigen-antibody interactions and optimizing antibody drugs.

These additional techniques mean Shuimu offers a comprehensive suite of structural determination options, ensuring the most appropriate method, or combination of methods, can be applied to a given project.

Case Sharing and Expertise

Shuimu's expertise is demonstrated through numerous successful projects and publications. Their Cryo-EM Center has resolved structures for over 400 clients since 2017, with over 300 proteins resolved below 3.5Å resolution and over 300 single particle projects completed.

Examples of structures resolved using Shuimu's platform and contributing to high-impact publications include:

• The cryo-EM structure of the human GluN1-GluN2A subtype NMDA receptor bound with different small molecules.
• The structure of the H1R and rGq complex, studying the activation mechanism of histamine receptors.
• Structures related to SARS-CoV-2 (ACE2-spike RBD complexes, neutralizing antibodies, subunit vaccines), GPCRs (histamine H1 receptor, bradykinin receptors, κ-opioid receptor, adhesion GPCRs, D1 dopamine receptor, serotonin receptors, somatostatin receptors, GPR75), ion channels (Nav1.7), transporters (ATP13A2, MRP4, SPCA1, ASCT2, SLC18A1, TMEM16A, SLC2A17), and other complexes (dsRNA processing, Casπ, DNA polymerase θ, GSDMB, AAA+ chaperone, PARP7, CTBP2, ACYL, AFG3L, HpreP, Lonp1, TMEM119, CD73, WRN, USP1, USP5, SMARCA4).

These cases highlight the platform's capability to handle diverse and challenging targets, including membrane proteins and complexes relevant to drug discovery. The development and application of advanced techniques like GraFuture™ contribute to the ability to tackle such complex structural problems and achieve high resolutions.

For specific details on how Shuimu BioSciences can support your structural biology research, including the use of advanced sample preparation methods like GraFuture™, please visit https://shuimububio.com/.

Conclusion

Conventional cryo-EM SPA is a powerful tool, but faces limitations, particularly in sample preparation, affecting resolution and success rates for challenging samples like those at low concentration, small molecular weight, or prone to preferred orientation. Shuimu BioSciences's GraFuture™ graphene support grids represent an advanced technique specifically designed to overcome these bottlenecks.

By potentially solving issues like preferred orientation and enabling the analysis of samples previously difficult to study, GraFuture™ enhances the efficiency and accuracy of cryo-EM structure determination. Combined with Shuimu's extensive instrument platform, expert team, proprietary AI software (SMART, eTasED), and comprehensive upstream protein services, these advanced techniques position Shuimu BioSciences as a leading provider for high-resolution structural biology research. Whether dealing with routine samples or highly challenging targets, Shuimu offers a one-stop solution to achieve high-resolution 3D structures.

To explore the potential of GraFuture™ and other advanced cryo-EM techniques for your research project, and to benefit from Shuimu BioSciences's expertise, visit https://shuimubio.com/ today.