Cryo-EM Protein-Small Molecule Complex Structure Characterization Service

In structure-driven drug discovery, understanding the binding modes between small molecule drugs and target proteins is crucial for optimizing lead compounds and designing novel molecules. Traditional structural biology techniques, such as X-ray crystallography and Nuclear Magnetic Resonance (NMR), have been widely applied to analyze protein-small molecule complexes. However, these methods often struggle with systems that are difficult to crystallize, highly flexible, or exhibit conformational heterogeneity.

 



Zhu, KF. et al. Mil Med Res. 2023.

Figure 1. Application of Cryo-EM in Structure-Based Drug Design (SBDD)

 

Cryo-Electron Microscopy (Cryo-EM), an advanced technique that allows high-resolution 3D structural determination without the need for crystallization, has shown significant advantages in resolving protein-small molecule complex structures in recent years. MtoZ Biolabs leverages an advanced Cryo-EM platform to offer the Cryo-EM Protein-Small Molecule Complex Structure Characterization Service, assisting pharmaceutical, biotechnology, and basic research clients in understanding drug-binding mechanisms, optimizing structural designs, and driving innovative drug development.

 

Technical Principles

Cryo-EM is an electron microscopy technique that observes the structures of biological macromolecules under near-native conditions. The sample is rapidly frozen in liquid ethane, preserving its natural hydration state and spatial conformation. Electron beams are then used to image the sample, collecting a large number of 2D images, which are computationally reconstructed into a 3D density map.

 

For protein-small molecule complexes, Cryo-EM is particularly suited for capturing stable conformations after macromolecules bind with small molecules. In recent years, advancements in sample preparation, imaging equipment, and image reconstruction algorithms have enabled Cryo-EM to resolve structures of systems as small as 100 kDa or even smaller, making it a promising tool for studying protein-small molecule interactions.

 

Analysis Workflow

MtoZ Biolabs employs a combination of standardized operations and customized strategies in the Cryo-EM structural analysis workflow to ensure efficient acquisition and accurate characterization of various complex types:

1. Sample Preparation and Evaluation

Clients provide protein-small molecule complexes or their precursors. We assist with basic quality control including concentration, purity, and homogeneity, and offer advice on complex assembly and binding verification when necessary.

 

2. Grid Preparation and Rapid Freezing

Automated equipment is used to prepare the cryo-grids, followed by rapid freezing to ensure that the sample is frozen under near-physiological conditions, preserving its natural conformation.

 

3. Data Collection

High-end Cryo-EM is employed for low-dose imaging, collecting high-quality micrographs while minimizing radiation damage to the sample.

 

4. Image Processing and 3D Reconstruction

Professional software is used for image processing, particle classification, and 3D density map reconstruction, resulting in high-resolution structural maps of the complex.

 

5. Structural Modeling and Analysis

On the obtained 3D density map, small molecule fitting and refinement are carried out using molecular modeling software, generating complete structural models for further analysis.

 

6. Result Delivery and Reporting

A comprehensive structural analysis report is provided, including 3D images, structural models (PDB format), binding site annotations, and related interpretations, supporting clients' research or submission needs.

 

Service Advantages

1. No Crystallization Required, Efficient Resolution: Cryo-EM overcomes the crystallization bottleneck, making it suitable for non-crystalline proteins and flexible complex systems.

2. Low-Dose Imaging, Preserving Conformation: The natural conformation of protein-small molecule complexes is preserved, ideal for studying dynamic processes and flexible regions.

3. High-Resolution Modeling Capability: Coupled with structural refinement techniques, Cryo-EM can accurately locate binding sites and surrounding interaction networks.

4. Experienced Structural Analysis Team: MtoZ Biolabs has extensive project experience in protein-small molecule analysis, ensuring scientifically reliable model construction.

5. Supporting Experimental Assistance: We offer necessary sample optimization, assembly verification, and other supporting methods to improve the success rate of structural analysis.

 

Applications

This service is widely applied in structural biology and drug discovery-related scenarios, including:

1. Target Validation: Confirming the binding of small molecules with candidate proteins and their binding modes, providing structural support for target feasibility.

2. Drug Design and Optimization: Using structural information for structure-activity relationship (SAR) analysis, lead compound optimization, and novel scaffold construction.

3. Mechanism of Action Research: Revealing the binding behaviors of small molecules, such as agonists, inhibitors, or allosteric modulators.

4. Resistance Mechanism Analysis: Comparing binding site structures of mutant proteins or different states to study mechanisms of drug resistance.

5. Complex System Analysis: Supporting the study of multi-domain proteins, small molecule-enzyme complexes, multi-ligand systems, and other complex structural systems.

 

Case Study

1. Cryo-EM Structure of Small-Molecule Agonist Bound Delta Opioid Receptor-Gi Complex Enables Discovery of Biased Compound

This study employed cryo-electron microscopy to determine the structure of the δ-opioid receptor (δOR)-Gi complex bound to the small-molecule agonist ADL5859, revealing the receptor's activation mechanism and signaling bias characteristics. By using chemical probes, the researchers identified key receptor–ligand interaction sites and discovered a biased signaling compound, ADL06. The study clarified the role of the β-arrestin pathway in analgesic tolerance and side effects, expanding our understanding of δOR signaling and its biological functions. Cryo-EM Protein-Small Molecule Complex Structure Characterization Service can be applied to structural studies of membrane-bound receptors bound to small-molecule agonists. It supports the identification of functional binding sites, elucidates conformational states associated with receptor activation, and facilitates research into the selective regulation of downstream signaling pathways.

 



Cheng, L. et al. Nat Commun. 2024.

Figure 2. The Architecture of ADL5859-Bound δOR-Gi Complex

 

2. Cryo-EM Structure Determination of Small Therapeutic Protein Targets at 3 Å-Resolution Using a Rigid Imaging Scaffold

This study successfully resolved high-resolution Cryo-EM structures of several low-molecular-weight proteins by employing a rigid protein cage scaffold, including the key cancer signaling protein KRAS and its mutants (e.g., G12C). The results demonstrate the potential of this strategy in overcoming resolution limitations associated with small proteins and reveal conformational changes in the G12C mutant upon binding with the inhibitor AMG510, offering structural insights for small-molecule drug design. Cryo-EM Protein-Small Molecule Complex Structure Characterization Service can be applied to investigate the binding patterns between small-molecule drugs and low-molecular-weight target proteins. By enabling conformational comparisons, it supports the analysis of mutation effects and guides drug optimization strategies, particularly for disease-related targets such as those in oncology.

 



Castells-Graells, R. et al. Proc Natl Acad Sci USA. 2023.

 Figure 3. Cryo-EM Structure of KRAS G12C-Bound to AMG510

 

FAQ

Q1: Which types of protein-small molecule complexes are suitable for Cryo-EM analysis?

A1: Cryo-EM is mainly suitable for stable complexes formed after protein binding with small molecules, especially those that are difficult to crystallize, contain flexible regions, or exhibit high heterogeneity.

 

Q2: Does the small molecule need to have a high binding affinity?

A2: Higher affinity helps improve complex stability and imaging success. However, crosslinking or multivalent strategies can also be used to enhance binding efficiency.

 

Q3: Does this service include protein-small molecule assembly?

A3: We offer technical advice and some experimental assistance, such as optimization of ratios and binding verification, but the client is responsible for confirming the stability of the complex.

 

Q4: What is the typical resolution for structural analysis?

A4: The resolution depends on sample quality, molecular weight, and structural complexity, typically ranging from 3-5 Å, with some systems achieving better than 3 Å.

 

Q5: How long does the service take?

A5: From sample receipt to result delivery, the typical timeframe is 6-10 weeks, with potentially longer periods for complex projects or those requiring sample optimization.

 

For more information on Cryo-EM Protein-Small Molecule Complex Structure Characterization Service or to receive a project evaluation, please contact MtoZ Biolabs' technical support team. We will provide you with professional guidance and efficient service.