Protein structure determination by electron diffraction of 3-dimensional protein microcrystals

Crystallographic methods for protein structure determination are well established and have provided valuable insight into the workings of the biological world. X-ray crystallography of three dimensional crystals has benefited from the development of high throughput screening methods, but the method still requires the production of large well ordered crystals. Crystal screening and optimization is an extremely time consuming bottleneck and many proteins, often of high biological interest, are simply not able to be crystallized. Electron crystallography of two dimensional crystals is a much more limited method applicable only to extremely thin two-dimensional crystals, usually composed of protein and lipid. This method has provided insight into the structures and protein-lipid interactions of some integral membrane proteins but is extremely low throughput and not broadly applicable. This work presents a new method for protein structure determination which is essentially a hybrid of these two methods. A focused electron beam is diffracted by very small three dimensional protein microcrystals allowing for diffraction data to be collected from crystals at least six orders of magnitude smaller than those used for traditional X-ray crystallography. This method allows data collection and subsequent structure determination using crystals that would be considered 'initial hits' in a crystallization screen and would normally require a great deal of optimization before yielding data for structure determination. As proof of principle the structure of hen egg white lysozyme determined to 3.0 A resolution by microcrystal electron diffraction is presented. The procedures for data collection are detailed as well as an explanation of new software developed for processing the resulting data and possible future refinements and improvements to the technique. With further refinement microcrystal electron diffraction may prove useful for many proteins that are currently considered intractable because of their failure to form large well ordered three dimensional crystals.