(6cm) Overcoming Barriers in Structural Biology Though Novel Method Development

The 3-dimensional structures of proteins and other biomolecules are critical to understanding the mechanisms of biological processes. By far the most successful method in structural biology for structure determination is X-ray crystallography, a fact highlighted by the over 88,000 protein and nucleic acid structures deposited in the Protein Data Bank. Despite its remarkable successes, X-ray crystallography suffers from two major bottlenecks: the relatively large quantity of biological material required to screen crystallization conditions, and the need to form large well-ordered crystals. This poster is a presentation of recent research on overcoming these two main roadblocks in order to facilitate the structural study of more difficult biological targets. 

The first part of the poster focuses on the strategies used to address the large quantities of protein necessary for structural studies. The project specifically concentrated on improving the overexpression of membrane proteins in Esherichia coli. Membrane proteins are extremely important therapeutic targets and hold potential for practical applications in bionanotechnology. However, low yields in normally robust expression hosts such as E. coli have hindered progress in our understanding of their structure and function. By engineering the protein folding pathways in E. coli, we showed the expression levels of several membrane proteins could be improved 3 to 7 fold. Additionally, expression vectors were improved by directed evolution, resulting in reduced expression-induced toxicity and enhanced functional membrane protein yields in E. coli

The second part of the poster details the progress made on overcoming the other major roadblock of protein crystallography: the formation of large well-ordered crystals.  For difficult proteins (e.g., membrane proteins and protein complexes), initial crystallization screens will often times only produce small microcrystals which, despite years of attempted optimization, will never grow to suitable sizes required by traditional X-ray crystallography. We demonstrated for the first time that it is possible to use electron crystallography to solve a protein structure from exceedingly small 3-dimensional microcrystals, orders of magnitude smaller than what is required for X-ray crystallography. The development of this new method, which we named ‘MicroED’, promises to have a large impact on structural biology by allowing structural data to be obtained from protein crystals previously thought to be unusable.