Cryo-Electron Microscopy
CryoEM-SPA

CryoEM single particle analysis (SPA) uses the electron scattering mechanism combined with cryogenic freezing, which significantly reduces high-energy electron beam damage to molecular structures, enabling near-native high-resolution structure determination of proteins without crystallization.

How Single-Particle Analysis Works

In cryo-EM SPA, tens of thousands of 2D projection images of individual protein particles are collected, computationally aligned, and averaged to reconstruct a high-resolution 3D density map. Unlike crystallography, no ordered crystal is needed — particles are imaged in their near-native hydrated state embedded in vitrified ice.

The process begins with negative-stain TEM for rapid quality assessment, followed by cryo-sample preparation and high-resolution data collection on a 300 kV FEG instrument. Data processing uses cryoSPARC or RELION to produce density maps, into which atomic models are then built and refined.

A key advantage is the ability to capture multiple conformational states within a single experiment — 3D classification separates heterogeneous particle populations into distinct structural states, providing a dynamic view of protein function that is inaccessible to crystallography.

The client provides purified protein samples directly, or specifies the target protein sequence for iCDMO to perform in-house expression and purification. Sample purity, concentration, and buffer conditions are assessed and confirmed before proceeding to the next stage.

Heavy metal staining (uranyl acetate or uranyl formate) is applied for rapid quality assessment. Micrographs are analyzed to evaluate particle homogeneity, dispersity, size, shape, and sample concentration — providing a fast go/no-go decision before cryo-sample preparation.

Using a Vitrobot Mark IV plunge-freezer, 3–5 µL of protein sample is applied to a glow-discharged grid. The sample is blotted to form a thin molecular layer and rapidly plunged into liquid ethane (−185°C), vitrifying the sample in amorphous ice to preserve the native protein structure and spatial distribution.

Grids are screened using a 200 kV FEG microscope to assess ice thickness, particle distribution, preferred orientation, and 2D class average quality. Preliminary data collection provides resolution estimates and guides optimization of grid conditions before high-resolution acquisition.

High-resolution data is collected on a 300 kV FEG cryo-electron microscope (Titan Krios) equipped with a direct electron detector (Falcon 4 or K3). Automated acquisition software (EPU, SerialEM) collects tens of thousands to millions of single-particle micrographs with high-throughput automation.

Raw micrograph movies are processed through a multi-step computational pipeline: motion correction, CTF estimation, particle picking (Topaz/crYOLO), 2D/3D classification, ab initio model generation, and iterative 3D refinement using cryoSPARC or RELION 4. TB-scale HPC infrastructure supports large-scale datasets.

The refined EM density map is used for atomic model building in Coot or ISOLDE. Initial models from AlphaFold2 or homology modeling are fitted into the density map, followed by real-space refinement in PHENIX. Model geometry and fit-to-map quality are validated using MolProbity and PHENIX reports.

The final atomic structure is analyzed for biological insights: binding interfaces, conformational changes, potential drug-binding pockets, and mechanistic interpretation. A comprehensive project report is delivered including the EM density map, atomic coordinates (PDB file), local resolution maps, FSC curves, and publication-quality ChimeraX/PyMOL figures.

MRGPRX2 – G Protein-Coupled Receptor Complex

MRGPRX2 (Mas-Related G Protein-Coupled Receptor X2) is a GPCR primarily expressed in skin mast cells. It is mainly involved in non-IgE-dependent mast cell activation, playing a key role in Type VII hypersensitivity reactions and pseudo-allergic drug responses.

Using cryo-EM single particle analysis, iCDMO determined the near-atomic resolution structure of the MRGPRX2 complex with its G protein partner. The structure revealed the transmembrane helix packing geometry, key ligand-binding residues in the orthosteric pocket, and a cryptic allosteric site — providing the molecular basis for mast cell activation and a structural template for drug safety profiling and therapeutic development.