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

Abstract

This article addresses recent developments in cryo-electron microscopy and single-particle reconstruction of macromolecules. With the advent of powerful classification techniques, it is now possible to extract and visualize multiple conformers contained within the same dataset. It is discussed how and to what extent this technique can be used in the study of the dynamics of molecular machines.

Keywords: conformational states; free-energy landscape; ribosome; single-molecule FRET.

Fig. 1

Schematic diagram of (one-dimensional) energy landscape. (It depicts either a representative slice through the conformational coordinate or an average of the conformational coordinate). Three local minima (“basins”) denoted by State 1, Intermediate, and State 2 are separated by maxima (“hills”). Sizable subpopulations of molecules will only be found in the basins (indicated by fill in each basin), signifying states of relative stability. Points TS1 and TS2 mark lowly populated labile transition states which interconvert to either of the adjacent more stable states.

Fig. 2

The dilemma posed by multiple pathways. (a) A two- (and higher-) dimensional energy landscape allows multiple pathways between two given states. (b) An example for ambiguities in constructing a (sequential) narrative connecting different structures. Three of many possible pathways are shown between two states designated as “beginning” and “end.” The individual density maps used to illustrate this idea are the result of classification applied to a large dataset of ribosomes in the pre-translocational state.

Fig. 2

The dilemma posed by multiple pathways. (a) A two- (and higher-) dimensional energy landscape allows multiple pathways between two given states. (b) An example for ambiguities in constructing a (sequential) narrative connecting different structures. Three of many possible pathways are shown between two states designated as “beginning” and “end.” The individual density maps used to illustrate this idea are the result of classification applied to a large dataset of ribosomes in the pre-translocational state.

Fig. 3

Illustration of the idea that continuity of features – here across the one-dimensional boundaries of a two-dimensional puzzle – is required to reconstruct the scene in its entirety. In contrast, no continuity exists among the reconstructions harvested from a cryo-EM sample containing a molecule in multiple states. (Photo credit Sylvia Wentzlau, http://www.webdesign-und-fotografie-leipzig.de)