Revealing nanoscale structure and interfaces of polymer and protein condensates via cryo-electron microscopy.

Abstract

Liquid-liquid phase separation of macromolecules is a ubiquitous process studied across biological and material sciences. In one instance, phase separation of proteins can be driven by low complexity intrinsically disordered regions that permit multivalent interactions between polypeptides. In synthetic systems, a complexation of oppositely charged polyelectrolytes or the immiscibility of two blocks can drive phase separation of polyelectrolytes and block copolymers respectively. The result of these processes are condensates that display liquid-like characteristics. The nature of these condensates has mainly been investigated through methods based on optical microscopy, such as assessing condensate morphology, fusion dynamics, and diffusion of molecules. Importantly, current methods used to study condensates cannot probe the nanoscale structure of the material. Here, we developed a method that applies cryo-electron microscopy (EM) to investigate the internal structure of condensates in vitro at the nanoscale. By bypassing the drying of material for conventional EM, we preserve the internal organization of molecules. We tested a range of material preparation protocols, including varying the temperature, concentration, and buffer conditions of the solution, along with vitrification procedures. We are able to produce both micron-sized spherical droplets and thin films of condensed material that are amenable to cryo-EM imaging. We applied this method to study condensates composed of intrinsically disordered proteins, polyelectrolytes and block copolymers. Our results reveal the internal organization of the different materials at the nanoscale. Furthermore, we relate the material properties of condensates composed of intrinsically disordered proteins to their nanoscale structure. We anticipate the use of cryo-EM beyond single particle analysis to understand phase separation of intrinsically disordered proteins and other macromolecules.