Cryo-Electron Tomography Based Characterization of Liquid-Liquid Phase Separation

The assembly of membrane-free compartments by liquid-liquid phase separation (LLPS) is thought to be the mechanism for the spatial and temporal organization of intracellular biomolecules. The function of such mesoscale assemblies, called biomolecular condensates, depends on a network of multivalent interactions between proteins, their structured and disordered structural domains, and often also nucleic acids. However, these interactions pose a particular challenge for structural analysis due to the lack of a fixed stoichiometry. Cryo-electron tomography (cryo-ET) provides structural information from the Å to nm length scale and can be used to study biomolecules in their natural cellular environment. Cryo-ET is thus able to obtain structural information at length scales that are intermediate between optical microscopy and more traditional structural biology techniques.

Customized Services

Our team of experts is very interested in the nature of condensates highlighted by polymorphic interaction networks, and is committed to selecting a tool suitable for molecular resolution imaging at wide spatial scales in order to examine morphology and fine molecular detail. Here, CD BioSciences offers the ideal cryo-ET technique to visualize the entire biomolecular cohesive network, allowing easy study of the 3D structure of such polymorphic interaction networks at nanometer resolution. Our cryo-ET has been successfully used to characterize the gel-like droplets formed by yeast Sup35.

In addition, we offer a focused ion beam (FIB) grinding method guided by an associated 3D fluorescence microscope that allows cryo-ET studies of specific regions within complex cell structures. Proudly, we are developing a series of protocols aimed at obtaining high quality cryo-electron tomography data of recombinant biomolecular condensates in vitro, including:

(1) Rigorous screening by light microscopy.

(2) Cryofixation by insert freezing.

(3) Sample loading into an electron microscope operating at liquid nitrogen temperature.

(4) Data collection.

(5) Processing of data into 3D tomograms and their interpretation.

Preparation of Suitable Condensate Cryo-Electron Microscopy Samples

• Must be retained stable on the electron microscope (EM) grid.
• Size must be strictly controlled.
• Maintain hydration.
• Strict sample thickness limits, <300 nm.

Advantages of Cryo-ET for Condensates Analysis

• Visualize the entire biomolecular cohesion network in three dimensions (3D).
• Has nanometer resolution.
• A label-free method that preserves the intrinsic molecular structure.
• Allows identification of detailed structural features in the condensate.

Based on our experience in imaging structures related to stress particles, centrosomes and P-particles, we provide detailed methods for the preparation of recombinant biomolecular condensates in vitro and 3D imaging using cryo-ET. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.