Characterization of Complexes and Supramolecular Structures by Electron Microscopy.

Recent advancements in cryo-electron microscopy (cryo-TEM) have enabled the determination of structures of macromolecular complexes at near-atomic resolution, establishing it as a pivotal tool in Structural Biology. This high resolution allows for the detection of ligands and substrates under physiological conditions. Enhancements in detectors and imaging devices, like phase plates, improve signal quality, facilitating the reconstruction of even smaller macromolecular complexes. The 100-kDa barrier has been surpassed, presenting new opportunities for pharmacological research and expanding the scope of crystallographic analyses in the pharmaceutical industry. Cryo-TEM produces vast data sets from minimal samples, and refined classification methods can identify different conformational states of macromolecular complexes, offering deeper insights into the functional characteristics of macromolecular systems. Additionally, cryo-TEM is paving the way for time-resolved microscopy, with rapid freezing techniques capturing snapshots of vital structural changes in biological complexes. Finally, in Structural Cell Biology, advanced cryo-TEM, through tomographic procedures, is revealing conformational changes related to the specific subcellular localization of macromolecular systems and their interactions within cells.

Observation of Bacteriophage Ultrastructure by Cryo-Electron Microscopy.

Transmission electron microscopy (TEM) is an ideal method to observe and determine the structure of bacteriophages. From early studies by negative staining to the present atomic structure models derived from cryo-TEM, bacteriophage detection, classification, and structure determination have been mostly done by electron microscopy. Although embedding in metal salts has been a routine method for virus observation for many years, the preservation of bacteriophages in a thin layer of fast frozen buffer has proven to be the most convenient preparation method for obtaining images using cryo-electron microscopy (cryo-EM). In this technique, frozen samples are observed at liquid nitrogen temperature, and the images are acquired using different recording media. The incorporation of direct electron detectors has been a fundamental step in achieving atomic resolution images of a number of viruses. These projection images can be numerically combined using different approaches to render a three-dimensional model of the virus. For those viral components exhibiting any symmetry, averaging can nowadays achieve atomic structures in most cases. Image processing methods have also evolved to improve the resolution in asymmetric viral components or regions showing different types of symmetries (symmetry mismatch).