Dynamic Structures and the Fourth Dimension in CryoEM

Many biological macromolecules can be envisaged as molecular motors, i.e., they employ biochemical energy to move other molecules in a specific manner and direction, tte link between reactions usually comes from conformational changes that macromolecules exploit to perform their tasks. In those cases, it is obvious that understanding the functioning requires following up those movements through the different steps of the process. What necessarily emerges is the addition of a fourth dimension, time, to 3D studies aimed at comprehending the course of action of large molecular assemblies. Cryo-EM is well suited for this purpose, since the structures are determined in near-native conditions and experiments can be designed to trap particular transient states. Here, the 3D exploration by cryo-EM at medium resolution provides a dynamic picture, and the aforementioned combination of the data with available atomic coordinates results in "quasi-atomic" structural models that reveal the biological mechanisms in detail. Since cryo-EM is an averaging technique, it is necessary to merge information coming from individual molecules that display the same conformation, tte idea is to capture the dynamic process through "snapshots," where each 3D map defines one stage of the reaction.

We can find different approaches in the literature that deal with the movement of macromolecules. tte straightforward option is to stop in vitro reactions while freezing the samples in liquid ethane, the so-called time-resolved cryo-EM. Bac-teriorhodopsin 2D crystals were analyzed as early as 1 ms after illumination to define light-driven changes in protein conformation (Subramaniam and Henderson

1999). tte feasibility of this technique, however, is currently limited by the nature of the agent that triggers the conformational change. Physicochemical factors, such as light, temperature and pH are easy to apply to the sample in a simultaneous and uniform way. In other cases, where additional molecules participate in the reaction, uneven diffusion and stochastic flows impede to trap a synchronized population of macromolecules.

Sometimes, the system can be slowed down, and the bottleneck created allows the desired synchronic state to be achieved. Following this strategy, the dynamics of herpes simplex virus maturation was studied after blocking the activity of a protease, since protease-deficient procapsids mature slowly in vitro (Heymann et al. 2003) (Fig. 10.4). Most dynamic structural studies by cryo-EM, however, employ biochemical interference that stalls the macromolecules in a desired step, tte ribosome is the paradigm of this type of characterization, since several natural antibiotics are known to inhibit translation, tte incorporation of specific transfer RNAs (tRNAs) to the ribosome was analyzed thanks to the blockade of the process by kirromycin (Valle et al. 2003a). tte described rearrangements of ribosome and tRNA uncovered a cascade of interrelated events that clarified the structural basis for fidelity during translation.

Even in cases where the reactions cannot be stopped at the desired step, cryo-EM offers another possibility. Some investigators, rather than synchronize the macromolecules during their activity, have tried to analyze a full dynamic reaction within the same unique sample. In this approach, a cycling reaction takes place in a tube where the molecules alternate between two or more conformations, resulting into a mixed data set. tte challenge consists in separating different homogeneous groups of individual particles while processing the whole population. Here, classification tools are indispensable in order to differentiate the variability of the images due to distinct orientations of the molecules from a genuine structural diversity among them. Few attempts have been made following this risky scheme, and in most of them, the success depends on the availability of templates the particles can be compared with. Promising results, however, have been presented with the ATP cycling of chaperonins (Schoehn et al. 2000) or the ratchetlike relative rotation between ribosomal subunits during translocation of tRNAs (Gao et al. 2004).

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