#cryoem

Bound for Life

DNA replication is crucial for life; without it we wouldn’t grow. A key step in replication is the formation of a fork. A replication fork can be compared to the zip on your jeans – the DNA helix is unwound by an enzyme, helicase, allowing other enzymes access to copy the DNA. However, how helicase binds to DNA was a mystery. Researchers at MRC LMS and Imperial College London discovered in yeast that helicase, made of six proteins called Mcm2–7, is loaded onto the DNA in four steps and undergoes structural changes throughout the process. Seen here in cryo-EM images are the first three steps (rows top to bottom) with helicase highlighted in green and the loading complex in blue around the DNA coloured red. DNA replication in yeast and humans is extremely similar so this research has the potential to be applied in healthcare, such as inhibiting DNA replication in cancer.

Read more about this research here

Written by Lauren Green

Image from work by Zuanning Yuana, Sarah Schneider, Thomas Dodd and Alberto Riera, and colleagues

DNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, UK

Image copyright held by the original authors

Research published in PNAS, July 2020

Science, Volume 389, Issue 6756, July 2025. Summary I cannot provide a summary because I do not have access to specific content from a future publication like “Science, Volume 389, Issue 6756, July 2025.” My knowledge is based on the information available up to my last knowledge update. To get a summary, you’ll need to consult the actual issue of Science when it is published. Read more… Credits:…

Understanding the intricate three-dimensional structures of biological molecules is fundamental to advancing research in life sciences and pharmaceutical development. Protein structure determination is particularly crucial, as proteins are the workhorses of the cell, involved in nearly every biological process. Knowledge of their structures provides critical insights into their functions, interactions, and how they can be targeted by drugs. While traditional methods like X-ray crystallography have been invaluable, techniques such as Cryo-Electron Microscopy (Cryo-EM) and Microcrystal Electron Diffraction (MicroED) have emerged as powerful tools, capable of resolving structures of molecules previously intractable to other methods, including complex protein assemblies and membrane proteins. These technologies offer unprecedented resolution and versatility, driving innovation in drug discovery, vaccine development, and fundamental biological research.

Cryo-EM, particularly Single Particle Analysis (SPA), is a robust method that combines cryogenic electron microscopy with sophisticated computer algorithms to determine the high-resolution three-dimensional structures of biological macromolecules. This technique involves imaging a large number of purified biomolecule particles in a vitrified (flash-frozen) state and reconstructing a 3D model from these 2D images.

Cryo-EM Single Particle Analysis (SPA) for Protein Structure Determination

Single Particle Analysis (SPA) using Cryo-EM is a powerful tool for resolving the high-resolution three-dimensional structures of biological macromolecules such as proteins and viruses. A key advantage of Cryo-EM SPA is its ability to preserve samples in a near-native state. It can also capture multiple conformational states of a molecule. The technique requires relatively small amounts of sample. Furthermore, it is effective in determining the structures of heterogeneous protein complexes.

Cryo-EM SPA is widely applied to resolve the 3D structures of biomacromolecules at high resolution. This includes various types of proteins such as membrane proteins (like GPCRs, ion channels, and transporters), enzymes, and ribosomes. It is also used for resolving the structures of DNA and RNA, including DNA's double helix and its interactions with other molecules, as well as viral RNA and ribosomal RNA (rRNA). Protein-nucleic acid complexes, such as transcription complexes and viral capsid protein-viral RNA complexes, can also be analyzed. Additionally, Cryo-EM is effective for studying the structures of virus particles, including SARS-CoV-2 (specifically its S protein complex with human ACE2), influenza virus (surface spike protein), African Swine Fever Virus (ASFV), Human Herpesvirus 6B (HHV-6B), and Rabies Virus Glycoprotein (VSV-GP).

The process for Cryo-EM SPA typically follows a detailed workflow: project consultation and communication, initial assessment, plan finalization, contract and payment, protein expression and purification, negative staining characterization, sample freezing and data collection, 2D particle picking, 3D structure reconstruction, model refinement, and final data delivery.

To enhance the efficiency and accuracy of structure determination, ShuimuBio offers specific advantages in Cryo-EM SPA. Their facilities include state-of-the-art Cryo-EM microscopes designed for high-quality structure determination, equipped with top-tier specifications and advanced computing platforms. The team is composed of doctoral-level scientists from leading institutions, specializing in structural biology, protein science, and computational biology. They have extensive experience, having completed over 200 Cryo-EM projects covering diverse areas from membrane proteins to antigen-antibody complexes. A core focus is achieving extreme resolution, with over 150 protein structures determined and a best resolution reaching 1.8 Angstroms. ShuimuBio utilizes an AI-driven platform, including independently developed SMART software, to improve the efficiency of Cryo-EM data analysis.

Challenges in traditional Cryo-EM sample preparation, such as air-liquid interface issues, severe preferred orientation, high sample concentration thresholds (>1μM), significant background noise, and difficulty reconstructing small macromolecules, can impact results. To address these, ShuimuBio has developed a series of graphene grid support films called GraFuture™. These include GraFuture™ GO (oxidized graphene oxide) and GraFuture™ RGO (reduced graphene oxide) support films, offering potential solutions for preferred orientation and improving results for samples with low molecular weight, low concentration, strong background noise, and issues caused by the air-liquid interface.

Cryo-EM is also used for Cryo-characterization, which involves observing and analyzing sample structures at ultra-low temperatures to maintain their natural state. This is particularly advantageous for observing the structures of proteins, liposomes, exomeres, and material interfaces. ShuimuBio specializes in the study of LNPs, liposomes, AAVs, and other viral vectors using this method. They utilize NanoSMART, their self-developed AI Cryo-EM system, which automatically identifies nanoparticle features from images from various electron microscopes, providing detailed reports with a single click. NanoSMART can also enhance the clarity of low-quality images for improved recognition. The system provides efficient and accurate characterization services, offering detailed reports covering size distribution, circularity, lamellar structure, fill/empty status, and integrity.

Negative staining is another valuable electron microscopy technique offered by ShuimuBio. Unlike methods that directly stain the sample, negative staining dyes the background, allowing the sample's structure and morphology to be clearly displayed while the sample itself remains unstained. This technique is widely used for observing viruses, nanoparticles, organelles, and other small structures. Negative Staining 2D refers to analyzing samples using two-dimensional images based on the negative staining method, particularly for characterizing structures arranged on a 2D plane, such as viruses or protein complexes. Negative staining, performed on a Talos L120C microscope, can provide preliminary information at a lower cost, including sample particle size, homogeneity, oligomeric state, morphology, particle density/sample concentration, protein structure, flexibility, sample integrity, conformation, and compositional heterogeneity. It's a useful step before Cryo-EM for preliminary quality assessment.

ShuimuBio's Cryo-EM platform has contributed to numerous research achievements published in top international journals, including atomic-resolution structures of ion channels, GPCRs, antigen-antibody complexes, and spliceosomes. Specific examples include the structure of human GluN1-GluN2A subtype NMDA receptors bound to different small molecules, the structure of the human histamine H1 receptor/Gq complex, and the structures of human bradykinin receptors. They have also determined structures of nanoparticle vaccines based on SARS-CoV-2 RBD and HR, neutralizing antibodies against SARS-CoV-2 variants, and a subunit vaccine candidate for COVID-19.

Applications of Cryo-EM in Protein Structure Determination

The applications of Cryo-EM in resolving protein structures are extensive, particularly in the pharmaceutical and life science industries.

In vaccine development, Cryo-EM plays a vital role by providing near-atomic resolution 3D structures of viruses. This helps researchers understand viral invasion mechanisms, which is crucial for vaccine design. Examples include resolving the structures of SARS-CoV-2 receptor ACE2 and its complex with the S protein, developing new strategies for live attenuated influenza vaccines by analyzing structures, and studying how neutralizing antibodies block measles virus infection. Cryo-EM is also used in vaccine quality control to assess the morphology, size, integrity, and aggregation status of vaccine particles at different production stages, aiding process optimization. It can detect the aggregation level of vaccine components, important for enhancing immune efficacy. Furthermore, Cryo-EM is used to study the interaction mechanisms between antibodies and vaccine antigens, helping to optimize vaccine immunogenicity. Examples include resolving the structures of vaccine-induced antibody complexes with HIV-1 Env protein and studying antibody binding mechanisms to influenza virus surface proteins. Cryo-EM is also critical for responding to virus mutations by quickly resolving the structures of new variants, helping scientists adjust vaccine design strategies.

In the field of antibody drugs, Cryo-EM is significantly valuable. It can resolve high-resolution 3D structures of antibody-antigen complexes, helping researchers understand antibody recognition mechanisms and binding sites. This structural information is essential for designing more effective antibody drugs. Cryo-EM is also used to study the mechanism of action of antibody drugs, including how they bind to targets and activate or inhibit signaling pathways. For instance, Cryo-EM was used to determine the structure of a broad-spectrum neutralizing bispecific antibody targeting SARS-CoV-2 Omicron variants, revealing the molecular basis of its activity and aiding the development of new antibody drugs against variants. The technique assists in optimizing and designing antibody drugs by revealing dynamic processes and conformational changes upon antibody-antigen binding, guiding the design of antibodies with higher affinity and specificity. It can also analyze antibody conformational epitopes, guiding antibody engineering. Membrane proteins, like GPCRs, are common targets for antibody drugs. Cryo-EM can resolve their high-resolution structures, revealing mechanisms of ligand binding, receptor activation, and signal transduction, providing crucial structural information for developing antibody drugs targeting these proteins. The speed of Cryo-EM data collection and structure resolution helps accelerate the antibody drug development process.

For small molecule drugs, Cryo-EM also has important applications. It resolves the high-resolution structures of biological macromolecules (such as membrane proteins and enzymes) that serve as drug targets, helping researchers understand where small molecules act. For example, determining GPCR structures using Cryo-EM allows detailed observation of GPCR binding with small molecule ligands, providing a structural basis for designing highly selective and effective small molecule drugs. Cryo-EM can study the interaction mechanisms between small molecule drugs and their targets. By determining the complex structures of GPCRs bound to small molecule agonists or antagonists, researchers can understand how drugs activate or inhibit receptors and regulate downstream signaling pathways, crucial for optimizing drug design and improving efficacy. Cryo-EM also shows significant potential in fragment-based drug discovery (FBDD) by revealing details of interactions between small molecule fragments and protein targets, assisting in screening and optimizing potential drug candidates. The high resolution and rapid data collection capabilities of Cryo-EM accelerate the small molecule drug development process by quickly providing detailed structural information, such as for GPCRs, to optimize drug design. Cryo-EM has unique advantages in studying biased ligands, which selectively activate or inhibit specific downstream signaling pathways mediated by GPCRs for more precise therapeutic effects. By determining the structures of biased ligands bound to GPCRs, their mechanism of action can be revealed, providing an important reference for developing novel small molecule drugs. Cryo-EM can resolve complex biomolecule structures, including membrane proteins and enzyme complexes, which are important targets in small molecule drug development.

MicroED for High-Resolution Structure Determination

Microcrystal Electron Diffraction (MicroED) is an cutting-edge technique capable of precisely resolving high-resolution structures from microcrystals and nanocrystals. It is particularly well-suited for organic compounds. ShuimuBio excels in using MicroED technology to provide precise structural insights for small molecule samples, peptides, and protein crystals. They offer free feasibility assessments and risk alerts, leveraging their extensive experience in electron microscopy and protein structure determination to help clients accurately evaluate their research goals.

ShuimuBio possesses the capability to use MicroED technology to provide high-resolution structures for challenging small molecule samples, peptides, and protein crystals. They have an original eTasED software that seamlessly integrates MicroED technology into conventional Cryo-EM systems without requiring any additional modifications, significantly boosting research efficiency and accuracy. The team consists of doctoral scientists from top institutions, proficient in both Cryo-EM and MicroED technologies. ShuimuBio boasts a high success rate, having successfully delivered over 80% of their MicroED projects, achieving resolutions between 0.6 and 1.0 Angstroms. Beyond small molecules, e-TasED can also resolve the structures of peptides and proteins. Examples of structures determined include Proteinase K (1.50 Angstrom, 29.05 kDa), FUS LC RAC1 (0.65 Angstrom, 0.66 kDa peptide), and Acetaminophen (0.65 Angstrom, 0.66 kDa peptide).

Integrated Protein Preparation and Analysis Services

Achieving high-quality structure determination, whether by Cryo-EM or MicroED, fundamentally relies on obtaining high-quality protein samples. ShuimuBio's protein platform is dedicated to preparing high-quality target proteins using three primary expression systems: bacterial, insect, and mammalian cells. They also utilize cell-free expression systems. Each system has its advantages and disadvantages depending on the target protein properties.

E. coli Expression System: Widely used and economical, offering rapid growth, high yield, and stability. It is particularly advantageous for small protein production but may result in inclusion bodies and lacks post-translational modifications.

Mammalian Cell Expression System: Preferred for producing therapeutic proteins, vaccines, and antibodies as it yields proteins closest to their natural state. Mammalian cells provide proper protein folding and post-translational modifications, resulting in recombinant proteins with molecular structures, physicochemical properties, and biological functions most similar to native higher organism proteins, increasing the likelihood of obtaining native biological activity.

Insect Cell Expression System: Uses baculovirus vectors for high expression efficiency and large foreign gene insertion capacity. It also performs post-translational modifications and processing, resulting in biologically active proteins.

Cell-Free Expression System: Synthesizes proteins in vitro using cell extracts, reducing the expression process from days to hours for a faster and more efficient overall process.

ShuimuBio also offers comprehensive protein purification services, including affinity chromatography, ion exchange chromatography, gel filtration chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC).

Protein characterization and quality control are vital. Services include assessing protein purity and homogeneity using methods like SDS-PAGE, BN-PAGE, and HPLC. Protein validation and modification analysis can be done via mass spectrometry. Protein-protein or protein-molecule binding analysis can be performed using techniques like SPR, BLI, MST, and ITC.

ShuimuBio has deep experience in the membrane protein field, with expertise in designing production and purification methods for membrane proteins, including GPCR, ion channel, and transporter sequences. They maintain a strict quality control system based on rigorous Cryo-EM analysis and characterization to ensure samples fully meet research requirements. They also offer a "shelf protein list" containing important drug targets, including many membrane proteins like GPR75, PolQ, and OX-2. Custom protein target services are also available.

Integrated "One-Stop" Solutions

ShuimuBio provides "one-stop" solutions for various structural biology needs. Their "one-stop" SPA solution covers a range of targets including antigen-antibody complexes, small molecule-target complexes, Protacs, membrane proteins (GPCRs, ion channels, transporters), VLPs, and peptides. Similarly, they offer "one-stop" crystal structure determination services for molecules like KRAS and SARS-CoV M protein. This integrated approach, combining protein preparation, purification, characterization, and structure determination technologies like Cryo-EM and MicroED, streamlines the research process, potentially saving time and costs for clients.

Why Choose ShuimuBio for High-Resolution Protein Structure Determination?

Choosing the right partner for complex scientific endeavors like protein structure determination is paramount. ShuimuBio stands out due to several key strengths:

Global Scale Leader: They operate the largest commercial Cryo-EM platform in Asia, equipped with eight 300 KV Cryo-EM units across Beijing and Hangzhou.

Extensive Experience: With experience from over 400 Cryo-EM projects, they have successfully determined over 150 protein structures.

Exceptional Resolution: They are committed to achieving the highest possible resolution, having reached a best resolution of 1.8 Angstroms and successfully determined the structure of proteins as small as 51kDa using Cryo-EM. With MicroED, they have reached resolutions of 0.6-1.0 Angstroms.

AI-Driven Innovation: Proprietary AI algorithms and software like SMART and NanoSMART significantly enhance data analysis efficiency and accuracy.

Proprietary Technologies: Development of GraFuture™ graphene grids addresses challenging sample preparation issues like preferred orientation. eTasED software enables MicroED on standard Cryo-EM systems.

Expert Team: Their core team consists of highly experienced scientists from top institutions.

Comprehensive Services: They offer "one-stop" solutions from gene sequence to high-precision 3D structure, including protein expression, purification, characterization, Cryo-EM, MicroED, and crystal structure determination.

Rigorous Quality Control: Emphasis is placed on quality throughout the process, particularly in protein sample preparation and data collection.

Efficient Operations: Offering 24/7 Cryo-EM machine time services with efficient booking and data collection.

These advantages enable ShuimuBio to provide high-resolution protein structure determination services efficiently and reliably, accelerating research and development across various life science disciplines.

Conclusion

High-resolution structure determination, particularly protein structure determination, is fundamental to unlocking the complexities of biological systems and developing next-generation therapeutics. Cryo-EM and MicroED have revolutionized this field, providing unprecedented access to detailed structural information for a wide range of biomolecules, including challenging targets like membrane proteins and small microcrystals.

ShuimuBio is at the forefront of this revolution, offering a comprehensive suite of services powered by state-of-the-art technology, scientific expertise, and innovative proprietary solutions. From high-quality protein preparation to advanced Cryo-EM SPA, Cryo-characterization, negative staining, MicroED, and crystal structure determination, they provide integrated workflows designed to meet the demanding needs of modern drug discovery and biological research. Their commitment to quality, resolution, and efficiency makes them a valuable partner in the pursuit of detailed molecular insights.

Chromatin remodeling plays a vital role in gene regulation, affecting how DNA is accessed. Disruptions in this process can also lead to cancer and other diseases. To better understand how chromatin remodeling works, scientists at St. Jude Children’s Research Hospital used cryo-electron microscopy (cryo-EM) to obtain fine structural details of a human chromatin remodeler in action. The researchers…

A collaboration between the U.S. National Committees on: Crystallography, Chemistry, CODATA & the Board on Chemical Sciences and Technology.

As part of Advancing Drug Discovery: A Webinar Series of the National Academies of Sciences, Engineering and Medicine, a session on Beyond the Microscope: Cryo-EM’s Impact on Accelerating Drug Discovery will be held on Thursday, November 16th, 2023 at noon (EDT). The 60-minute session will consist a presentation from Sandra Gabelli, Head of Structural biology at Merck & co and formally Executive Director of Protein and Structural Chemistry.

Over the past decade, single particle cryo electron microscopy (cryoEM) has transitioned from a niche technique to a powerful tool for structural biology. This is due, in part, to technological breakthroughs which have made it relatively routine to achieve near molecule formulations for protein targets of pharmacological interest in complex with drug candidates. The pharmaceutical industry has historically relied heavily on X-ray crystallography to enable structure-based drug design (SBDD); however, today cryoEM is playing an ever-increasing role in that process, especially for challenging samples like large multimeric complexes, proteins that are flexible, or proteins that are difficult to express or purify. Dr. Gabelli will describe the strategy and present examples were cryoEM has impacted the pipeline at Merck by enabling biology, guiding SBDD, driving protein engineering of biologics and impacting small molecule formulations with micro ED. Dr. Gabelli will also explore the potential offered by upcoming technological advancements of the technique.Register