Story in a sample-the potential (and limitations) of cryo-electron microscopy applied to molecular machines.

Molecular machines are workshops in the cell that bring molecules and substrates together in a coordinated, processive way.1–2. The molecular complexes acting as molecular machines are often spatially localized and assembled in an ad-hoc fashion, in response to the demands of the cell’s metabolism. Products of such binding reactions can be RNA or DNA polymers, ATP, or other compounds needed at the site. Well-known examples are RNA and DNA polymerases, ATP synthase, the ribosome, and the proteasome. Visualizing molecular machines in their various states of processing poses challenges which traditional techniques of structure research find hard to meet. The complexes formed in the course of the work cycle are typically large, relatively unstable, and flexible -- all properties that disfavor X-ray crystallography as a means of visualization. In addition, molecular machines can have multiple binding partners and can go through numerous states, characterized by different conformations and binding configurations. Purification of such complexes can be done through two different routes: either from a cell extract or from an in vitro reconstituted system. In either case, it is difficult to isolate the complex by biochemical means in one of its many states with the purity required for structural research. Cryo-electron microscopy (cryo-EM) is a fairly new technique for obtaining a 3D image of a molecule from a large set of projections, harvested from micrographs of a field of molecules captured in random orientations.3 In some cases, resolutions in the range of 4 to 5.5 A have already been achieved for the ribosome, an RNA-containing molecular machine.4–6 Purity of the sample in terms of conformation and binding state of the molecule is evidently an absolute requirement for obtaining a meaningful 3D image from the “single particle” projection data set. In certain cases, high purity can be achieved by affinity imaging techniques.7–9 However, because of inherent difficulties of finding biochemical methods that guarantee 100% purity, it has been a goal in the development of cryo-EM to accomplish the purification of the molecules after the imaging, at the data processing step. Cryo-EM projection data have a typical signal-to-noise ratio in the order of 0.1 or even less.10 The challenge posed by classification of such noisy data is compounded by the fact that variability stemming from a change in viewing direction is intermingled with variability due to heterogeneity of composition or conformation. Due to recent advances in cryo-EM, particularly in the methods of data analysis, it has indeed recently become possible to perform the post-imaging purification of a heterogeneous sample: by using new methods of classification, images can be divided into homogeneous classes, and thus separate reconstructions can be obtained for each of the states of a molecular machine. Since an entire spectrum of states becomes observable in a single sample, all connected by pathways along which the molecule’s trajectory unfolds, we may figuratively speak of a “Story in the Sample.”11 The following considerations were inspired by recent results in the study of the ribosome by both cryo-EM and single-molecule fluorescence resonance energy transfer techniques (smFRET),12–14 which invite a generalization to other systems, and lead to an appreciation of the potential these new techniques will have in exploring functionally relevant states of molecular machines. Cryo-EM shares with smFRET the unique property that it allows tracking of the signal coming from a single molecule and at the same time gives robust ensemble averages from thousands of molecules tracked. There is a sense that cryo-EM as a technique of structural biology is at an important juncture where application of concepts of statistical mechanics has become feasible and in all likelihood quite productive.15