While the revolution will not be crystallized, biochemistry reigns supreme

Abstract

Single-particle cryo-electron microscopy (EM) is currently gaining attention for the ability to calculate structures that reach sub-5 Å resolutions; however, the technique is more than just an alternative approach to X-ray crystallography. Molecular machines work via dynamic conformational changes, making structural flexibility the hallmark of function. While the dynamic regions in molecules are essential, they are also the most challenging to structurally characterize. Single-particle EM has the distinct advantage of being able to directly visualize purified molecules without the formation of ordered arrays of molecules locked into identical conformations. Additionally, structures determined using single-particle EM can span resolution ranges from very low-to atomic-levels (>30-1.8 Å ), sometimes even in the same structure. The ability to accommodate various resolutions gives single-particle EM the unique capacity to structurally characterize dynamic regions of biological molecules, thereby contributing essential structural information needed for the development of molecular models that explain function. Further, many important molecular machines are intrinsically dynamic and compositionally heterogeneous. Structures of these complexes may never reach sub-5 Å resolutions due to this flexibility required for function. Thus, the biochemical quality of the sample, as well as, the calculation and interpretation of low-to mid-resolution cryo-EM structures (30-8 Å ) remains critical for generating insights into the architecture of many challenging biological samples that cannot be visualized using alternative techniques.

The Central Role of the Tail in Switching Off Myosin II in Cells

“…3D, E, F, Movie 2), in contrast to earlier studies where the tail could not be identified [1] or was not analyzed in 3D [9]. These earlier studies illustrate the difficulties of visualizing the myosin tail by cryo-EM and the ease with which it can be seen by negative staining, our technique of choice (see [33, 34] and Methods). While the resolution of the reconstruction is not high (due partly to flexibility of the molecule), we could easily detect the different domains of the myosin heavy chain and light chains, revealing steric blocking of specific regions of the heads by the tail that explains how the molecule is so effectively switched off in the quiescent state [31].…”

“…Turkey gizzard smooth muscle myosin at physiological ionic strength (0.15 M NaAc) in the presence of ATP was observed by negative staining with 1% uranyl acetate, a technique providing key insights into protein domain structures and interactions [33, 34]. The compact, folded (10S) conformation dominated the EM images, with a small number of antiparallel 10S dimers, displaying a dumbbell-shaped structure (Fig.…”

“…The fit implies excellent preservation of the IHM in negatively stained single molecules ( cf . [33]), without significant flattening, and enables us to interpret head structure and interactions at higher resolution (<7 Å) than the reconstruction itself [37].…”

“…Smooth muscle myosin has similar structural and functional properties to nonmuscle myosin II and is a convenient source for our experiments on the 10S structure [10]. Myosin molecules in 0.15 M NaAc, 1 mM EGTA, 2.5 mM MgCl 2 , 0.5 mM ATP, 10 mM MOPS, pH 7.5 were negatively stained at 10 nM concentration using 1% (w/v) uranyl acetate [33]. Negative staining is a simple and powerful technique for obtaining 3D structural data at moderate resolution.…”

Cryo-EM: beyond the microscope

“…For example, during the process of specimen preparation, where a thin aqueous suspension is created and plunge-frozen, functionally relevant protein complexes that are stable in bulk solution may become dissociated [15-17]. In addition, proteins and protein complexes in aqueous solution are intrinsically flexible to varying extents [18], creating structural heterogeneity that can be even more pronounced in multi-domain proteins and multi-protein complexes [19,20].…”