3-D Electron Microscopy Group
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 of 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 but current development is concentrated on the first mode: a Microfluidic Mixer-Sprayer
Microfluidic Mixer-Sprayer. -- A novel integrated microfluidic device has recently been developed that allows small volumes of two reactants to be mixed in less than 1 ms, react for a time set by the device, and then sprayed onto a dry EM grid as it is being plunged into cryogen. Several devices have been constructed that allow reaction times ranging from 10 – 500 ms to be investigated. The current configurations require about 10 microliters of each macromolecular reactant per grid at a concentration in the micromolar range. The figure shows the mixer and sprayer components of the device (top left, middle left), the complete device (bottom left), the plumbed device mounted for operation (top right), and an example of a microspray plume directed towards a specimen grid (bottom right).
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.
Overview in the top panel shows the three syringe pumps (P). Left lower panel shows the grid (G) held by forceps (F) adjacent to the nozzle (N) that dispenses the pre-mixed solution. The filter-paper blotters (B) contact the grid for a set duration (right panel), after which the grid is plunged into the liquid ethane (E).
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.
References
- Lu Z , McMahon J, Mohammed H, Barnard D, Shaikh T R, Mannella C A, Wagenknecht T, T-M Lu T-M. (2009). Passive microfluidic devices for sub millisecond chaotic mixing. Sensors and Actuators B: Chemical (in press)
- Lu Z, Shaikh T R, Barnard D, Meng X, Mohamed H, Yasin A, Mannella C A, Agrawal R K, T-M Lu T-M, Wagenknecht T. (2009). Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy. J. Struct Biol. Aug 14. (Epub ahead of print) PubMed
- Barnard D., Lu Z., Shaikh T.R., Yassin A., Mohamed H., Agrawal R., Lu T.-M., and Wagenknecht T. (2009) Time Resolved Cryo-Electron Microscopy Of Ribosome Assembly using Microfluidic Mixing. Microscopy and Microanalysis 15: 942-943 Cambridge University Press doi:10.1017/S1431927609096925