Single Particle EM

Abstract

Single particle electron microscopy (EM) plays an important role in structural biology because it allows derivation of biologically relevant information about proteins and macromolecular complexes. A large amount of randomly oriented images of the specimen under study (so‐called particles) are collected from micrographs taken with an electron microscope. These particles are then computationally aligned and combined to yield the 3D structure, which is subsequently subjected to visualisation and interpretation. In most cases, the resolution attained with this technique precludes tracing of the polypeptide chain or the clear visualisation of the secondary structure elements. Nevertheless, the integrative combination of the information provided by the different structural techniques (X‐ray crystallography, EM, etc.) at different resolution levels has allowed a comprehensive interpretation of the structure. The recent advancements in instrumentation and computational procedures are now making it possible to obtain maps at sub‐nanometre, and even near‐atomic, resolution. Key Concepts Single particle electron microscopy (EM) allows the structural determination of proteins and macromolecular complexes. The combination of the structure obtained by single particle EM and the high‐resolution information obtained by other structural techniques allows the derivation of biologically relevant information. Structure determination at sub‐nanometre, and even near‐atomic, resolution is also being possible in a growing number of cases. The biological material has to be specially prepared prior to be imaged in the electron microscope. The 3D structure of the specimen is determined by collecting, aligning and combining a large number of randomly oriented particles of the specimen that are extracted from the microscope images.