Some of you must be curious about the science behind protein crystallization. Well, to the astonishment of many novices in the field, protein crystallization is an empirical science, even after so many years of research and no complete knowledge of protein crystallization exist. It does sometimes can be more art than science which demands minute observational capabilities, experience, persistence and gut feelings. These abilities and the knowledge of your protein’s characteristics will get you closer to crystallizing it much faster than a library full of text books on crystallography.
Even though there is no comprehensive theory behind protein crystallization, there are certain rules and physio-chemical behaviors which govern the basics of crystallization and without keeping them your success chances are smaller than before. I will share with you these basics though I truly recommend you checking the resources at the end of this post for more detailed explanations .
Supersaturation, nucleation and growth
The process of protein crystallization passes through two distinctive states
- Nucleation – the process in which several thousands of disordered protein molecules attach one to another to form a nucleus of orderly-packed nanometers-size aggregate. This is a crucial step in every crystallization event since this is the seed that will potentially generate a protein crystal.
- Growth – This process requires a nucleation event to precede, yet if the conditions are not fit, crystal growth will cease at the nucleation event and no crystals will be visible.
Nucleation requires high concentration of protein solutions or super saturation. How can we reach such high concentration such that we generate nuclei?
In most cases if you concentrate your protein to high concentration you will most probably notice that at a certain point white fluffy speckles will appear in the solution (or a brownish pellet), the unmistakable hallmark of (unordered) protein aggregation. There are also the lucky cases in which protein biochemists and crystallographers have found glistening (befringe) microcrystals floating in the protein solution while performing ultrafiltration. Unfortunately, such cases are terribly rare (though worth the venture once).
Since a brown pellet of unordered aggregation will not generate a diffraction pattern under the X-Ray beam (making it worthless in term of crystallography), we need to push the soluble protein to pack into ordered nucleation centers that will eventually form a crystal instead to collide and bind one to another without any repetitive order. This is the place at which crystallization methodologies come into play such as these (adopted from Table 1, McPherson 2004):
- Mixing protein solution with a crystallization solution to generate a supersaturation condition.
- Altering temperature, ionic strength and pH.
- Adding a ligand that affects the solubility of the protein.
- Dehydration of the protein solution.
- Addition of polymer to generate volume exclusion.
The above methodologies have a common starting point: a soluble protein solution which its solubility is gradually lowered such that neighboring protein units interact one with another to form an aggregate. One of the common ways to describe the path by which a protein crystallizes is the phase diagram. Phase diagrams are commonly plotted protein concentration vs. crystallization/buffer solution and is a simplified way to portray the behavior of the protein within the crystallization drop:
From this curve it is clear that using too high protein concentration might lead to precipitation (unordered aggregation) while using low concentration of protein solution might not reach the metastable region, at which crystallization occur. Since there are several crystallization methods, the behavior of the protein in initial experimentation can hint toward the preferred method:
- Vapor diffusion – In this method a mixture of protein solution and crystallization solution are placed in a sealed well which contains a reservoir of crystallization solution at higher concentration than that of the drop. Since this is a close system, water will diffuse via vapor from the drop to the crystallization reservoir, increasing the concentration of both protein solution and crystallization solution. Two types of vapor diffusion setups exists, most commonly placed in a 24 or 96-well plates matrix arrangement:
- Hanging drop – this is the classic crystallization method. Protein solution is pipetted at few microlitters volume on a round glass slide and commonly mixed with equal volume of crystallization solution (making the “crystallization drop” at 1:1 dilution) after which the slide is turned around and placed on top of a well pre-filled with the same crystallization solution. Thus, the crystallization drop faces or hangs up-side-down from the slide downward toward the bottom of the well (see figure 2A). Sealing of the system is done by waxing the well’s rim such that the glass slide sticks to the well and closes the system.
- Sitting drop – In this modern method, the same setup of the previous method is complemented by a stage platform on which the experimenter can place the crystallization drop (instead of placing it on a glass slide). Sealing is usually performed with a clear adhesive tape which seals the whole plate.
- Batch mode – Unlike vapor diffusion, in batch mode the protein solution is simply mixed with the crystallization solution and placed within a sealed system. The simple and relatively quick setup can be used when the crystallization conditions are known, when large volumes of solution are needed to be mixed (several hundreds of microlitters) or when the experimenter want to have quick assessment of the protein’s solubility limits. Microbatch is a sibling method to the batch mode which takes advantage of inert oil as a crystallization-supporting environment. The crystallization drop, which can be as small as a microlitter, is placed under oil (commonly paraffin oil) with no sealing thus enabling the gradual dehydration of water molecules from the crystallization drop, driving the concentration of protein and crystallization agents upward until complete dehydration occurs (thus enable to test the crystallization of undersaturated protein solution).
Which method to use? It depends on the case at hand:
- If you have copious amounts of protein at high concentration than vapor diffusion is the way to go. Many crystallographers use sitting drop as it is easier to setup, especially if you’re screening hundreds and thousands of crystallization conditions (try to imagine how much time it takes to wax each well and flip a slide with a crystallization drop on top).
- In cases where it is hard to observe crystals through the crystallization drop (if it is obscured by precipitations) then hanging drop is a better choice.
- In case you have a low concentration of your protein or limited amounts of your protein, you might consider using microbatch as it enable you to use relatively lower protein concentrations (<5mg/ml) and still obtain crystal hits. Since the crystallization drop is not exposed to air, microbatch can deliver different crystallization results and might even be the only way to get crystals so it is worthwhile to explore this technique in parallel to vapor diffusion experiments. You should note, though, that the microbatch crystallization will run until the drop is dried up (or until you seal the well) thus monitoring for the crystal formation should be conducted more frequently in comparison to the other methods.
In the next post I will describe how to setup your first crystallization plate.
- Protein Crystallization, Second Edition (IUL Biotechnology Series) by Terese Bergfors – an excellent hands-on book focused on protein crystallization.
- Outline of Crystallography for Biologists by D. M. Blow – A concise and simplified introduction to protein crystallography.
- Crystallography Made Crystal Clear, Third Edition: A Guide for Users of Macromolecular Models (Complementary Science) by Gale Rhodes – an in-depth text book dealing with protein crystallography.
- Protein crystallization explained by Bernard Rupp – an in-depth explanation of protein crystallization process