Protein Crystallization: The goal of the human genome project was to sequence the genes that code the approximately 30,000 proteins that comprise the human body. While much valuable information is contained in a protein's genetic code, it is often desirable to obtain the three-dimensional structure of the protein in order to elucidate its function. The first step in structure determination is obtaining purified protein, either directly through biochemical separation processes, or if the DNA sequence is known, from expression of the protein using genetically modified organisms. The next step is crystallizing the protein, and the final step is solving the protein structure from x-ray diffraction measurements. However, the structures of only 4,000 of the 30,000 human proteins have been solved, and these represent fewer than 300 of the estimated 1,000 unique protein folds. Most of these structures belong to compact, water soluble, single proteins. More problematic is the lack of human membrane protein structures. Only a handful of the 10,000 human membrane proteins have been solved! Another category for which there are only a few structures are protein complexes. On average, each protein strongly interacts with several other proteins. Some important protein structures, such as the ribosome and slicesome are composed of over 100 individual proteins. 

Video: Droplet generation using a coflow microfluidic device; the protein and precipitant are mixed on-chip to avoid any nucleation before starting the experiment. The stream labeled “Protein” contains lysozyme, 12.5% w/v PEG 8kD, and 0.1 M NaAc at pH 4.8, and the stream labeled “Precipitant” contains 12.5% w/v PEG 8kD, 10% w/v NaCl, and 0.1 M NaAc at pH 4.8. Videos: Sathish Akella. FradenLab YouTube.   


The rate-limiting step is the crystallization process. Crystallographers follow a set of recipes, which are the result of years of experience, and although intuition gleaned from colloidal chemistry aids in improvisation the crystallization process is poorly understood. A typical crystallography lab contains cold rooms stacked floor-to-ceiling with racks upon racks of suspensions of proteins in a range of solvent conditions. A theoretical framework for protein crystallization is needed in order to restrict the parameter space and eliminate crystallization as a bottleneck in structural determination of proteins. The current paradigm is that crystallization proceeds through nucleation and growth, but the microscopic details remain unclear.


Video: Lysosyme crystallization in emulsion droplets of volume of 1 nL produced using microfluidics. The crystallization conditions are 30 mg / mL lysozyme, 12.5% w/v PEG 8kD, and 5% NaCl in 0.1 M NaAc buffer at pH 4.8 and at 6 °C. The total duration of the crystallization trial is approximately 36 h. The stochastic nature of crystal appearance is a characteristic of an activated process. The number of drops without crystals as a function of time provides information about the nucleation rate. Videos: Sathish Akella. FradenLab YouTube.   


The theme running through our technology and methodology development research is the long recognized fact that the key to optimizing crystallization is the separation of nucleation and growth. By squarely confronting the fundamental kinetic nature of crystallization, we are developing complementary, portable, scalable and economical high-throughput technologies designed to exploit the kinetics of crystallization. One consequence of the particulars of our approach is that the crystals produced will be small. Independently of our technology, it turns out that for the other popular methods of crystallization, i.e. vapor diffusion and microbatch, small crystals appear more frequently than large ones. Our strategy is to exploit the relative abundance of small crystals over large ones by developing a technology to obtain structures from microcrystals.


Emulsion-Based Technique To Measure Protein Crystal Nucleation Rates of Lysozyme

Crystal Growth & Design 14, 4487-4509 (2014); Movies: DOI: 10.1021/cg500562r.


Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction 

IUCrJ 1, 349–360 (2014);  doi:10.1107/S2052252514016960