Faculty of Biological Sciences

Muscle Research

Millisecond Time Resolution Cryo-EM

The essence of any biological reaction is controlled or directed structural change [1]. Thus if we are to understand molecular mechanisms in detail, key reaction intermediates must be identified and characterised. Freezing is an excellent way to trap such intermediates, since cooling rates ~10exp6°C/sec are routinely attainable in small specimens. This results in vitreous rather than crystalline water. However, although freezing can be very fast, it was only comparatively recently that rapid mixing methods compatible with the thin specimens necessary for TEM have become available. A general method do this was developed by Berriman and Unwin [2] and involves a microscope grid layered with one reactant being sprayed with a second reactant just before freezing. The effective time resolution is the interval between the arrival of the spray and freezing, currently ~5 msec. This method is attractive because of its simplicity and wide applicability. Thus a range of reactants from small molecules to proteins and even protein assemblies can be delivered as aerosols. In a collaboration with Dr Howard White (Eastern Virginia Medical School), we have modified this approach to use computer control and rapid pre-mixing of the spray.

The animation shows the principle of the method. A grid covered with one reactant is blotted to a thin layer (<100 nm) and sprayed with the second reactant just before the grid is frozen by plunging into liquid ethane. A double syringe mixer allows the spray itself to be mixed just before use.


The Mark II versionThe Mark I apparatus blotted and plunged with pneumatic pistons. The Mark II version uses stepper motors (see picture). References 3-5 describe our apparatus and its use.


References


1. Moffat, K., and Henderson, R. (1995). Freeze Trapping of Reaction Intermediates. Current Opinion In Structural Biology 5, 656-663.

2. Berriman, J., and Unwin, N. (1994). Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicrosc. 56, 241-252.

3. Walker, M., Trinick, J., and White, H. (1995). Millisecond Time Resolution Electron Cryo-microscopy of the M-ATP Transient Kinetic State of the Acto-Myosin ATPase. Biophysical Journal 68, 87s-91s.

4. Walker, M., Zhang, X. Z., Jiang, W., Trinick, J., and White, H. D. (1999). Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry. Proc. Nat. Acad. Sci. (USA) 96, 465-470.

5. White, H. D., Walker, M. L., and Trinick, J. (1998). A computer controlled spray-freezing apparatus for millisecond time-resolution electron cryo-microscopy. J. Struct. Biol. 121, 306-313.

6. White, H. D., K. Thirumurugan, M. L. Walker, and J. Trinick. 2003. A Second Generation Apparatus for Time-Resolved Electron Cryo-microscopy Using Stepper Motors and Electrospray. J. Struct. Biol., 144:246-252.