Single particle electron cryomicroscopy: trends, issues and future perspective

Abstract

Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition

“…Novel structures of such intermediates are required to complete our understanding of the transition pathway for each class of viral fusion glycoproteins. Cryo-EM and cryo-ET enhanced by direct electron detection devices, improved microscopes with more stable optics, and advances in image processing software [88][89][90][91] should make it possible to visualize the conformation of the glycoproteins while they interact with a target membrane. Indeed, as mentioned above, some progresses have already been made for both class I [81][82][83][84]92] and class II fusion glycoproteins [67][68][69]; however, a higher resolution will be required to get a reliable quasi-atomic model of those intermediates.…”

Rational Design of Semi‐Synthetic Protein Complexes with the Defined Oligomeric State

“…The study of highly challenging homomeric and heteromeric protein complexes has been facilitated by a tremendous advancement in the area of cryo‐electron microscopy in the last decade . These naturally occurring protein complexes (PCs) serve as an inspiration for the design of synthetic as well as semi‐synthetic protein complexes through the bottom‐up approach.…”

Transporters Through the Looking Glass: An Insight into the Mechanisms of Ion-Coupled Transport and Methods That Help Reveal Them

“…Since most ion-coupled transporters are in the size range of 40-60kDa, cryoEM is still in a state where high resolution structural information is difficult to attain. However, novel EM tools like phase plates, and biochemical tools like antibodies that can enhance the size and reduce the conformational heterogeneity, will likely aid in structure determination of transporters through cryoEM in the near future (Vinothkumar and Henderson 2016). Given the important roles that the transporters play in physiology and disease and wide array of methodologies available to characterize them, research in ion-coupled transporters is primed for rapid expansion in the near future.…”