Resource for the Visualization of Biological Complexity (RVBC)

Time-resolved cryo-electron microscopy

Development of methods for studying macromolecular dynamics and interactions:

The goal of structural biology is to understand physiological function from three-dimensional organization. Electron microscopy (EM) is one general technique to obtain a 3D structure. Whereas the end product conventional EM is a static structure, it would be informative to observe a structure in a sequence of states in order to undertand how it works. Our goal is to develop methods to initiate reactions involving macromolecules, trap them in intermediate states, and visualize them using electron microscopy. We have implemented three modes to prepare time-resolved samples for EM:

Pre-mix -- The pre-mix mode involves delivery by micro-syringes of two sets of reactants to a mixing chamber -- similar to a stopped- or continuous-flow apparatus -- and then, after a pre-set time interval, depositing the mixture onto a grid, blotting, and then freezing. This method is the simplest to implement, but it has the poorest time resolution: the shortest time from mixing to freezing is between 1 and 2 seconds; the blotting step here is rate-limiting.

Electrospray -- With the spray method, microdroplets containing one reactant are sprayed onto an EM grid containing the other reactant. The grid here is blotted before spraying, with circumvents the rate-limiting step in the pre-mix mode. To achieve an even distribution of droplets and to minimize extra thickness added to the aqueous film on the grid, it would be desireable to keep the droplets small, on the order of one micron in diameter, and dense. To this end, instead of using the more classical atomizing spray, we have implemented an electrospray, of the type used in mass spectrometry.

Flash photolysis -- There exist chemical reagents that become able to react only upon irradiation with light. We have mounted a light pipe leading from a xenon lamp, trained on a point in the path of the grid as it is cryofixed. The two reactants of interests -- one in a unreactive, "caged" form -- are both on the grid at the start of the experiment. The grid is blotted (before the reaction has started) and then flashed. The length of time between flashing and cryo-fixation can be adjusted by the user.

Concurrent with these sample-preparation methods are computational techniques to distinguish between what are likely to be heterogeneous states appearing in images.

We have several in-house projects to test the above methods, including the ryanodine receptor with calcium, the ribosome release complex with release factor-1, and various ribosomal complexes with GTP.

Time-resolved apparatus