March 3, 2010 03:52
Most of us protein crystallizers are not physical chemists and are sometimes bewildered when looking at a protein crystallization phase diagram. The thing is, that phase diagrams serve as a useful conceptual framework and they can be used to guide the trouble-shooting process for protein crystallization trials.
So, here's it: Protein crystallization can be seen as three distinct events: nucleation, growth and stop of growth. Disregard the kinetics of these processes for simplicity sake. Crystallization occurs only from supersaturated solutions where the protein concentration exceeds its solubility in a given solution. The state of this supersaturation depends, of course, on many factors - tweaking these factors is the game we play in protein crystallization (we win when crystal nuclei form and crystals to grow).
If you haven't done so already, please take some quality time to take in this schematic protein crystallization phase diagram:
Figure: Protein crystallization phase diagram schema. Crystal nucleation is a critical phenomenon that occurs only in an area of the supersaturation zone. Crystals grow under conditions of supersaturation only once nuclei have formed. Yellow: no crystal growth; red: no crystal growth either. Dark-blue: this is the target area to land in for crystallization screening. Light-blue: this is where you want to be for crystal growth.
Once you're familiar with the lines separating the differenct zones, consider these three pathways:
Pathway A: batch-type crystallization. By mixing protein with the precipitant solution the protein becomes supersaturated. Crystal nuclei form and crystal grow until the protein concentration in solution is saturated.
Pathway B: vapor diffusion-type crystallization. The concentration occurs during vapor diffusion, following mixing of the protein with the precipitant solution causing the protein to become supersaturated. During vapor diffusion the precipitant concentration increases and extends the crystal growth process.
Pathway C: dialysis-type crystallization. As the precipitant diffuses into the protein-holding chamber the protein supersaturates. Once nuclei have formed, protein crystals grow as long as the protein concentration remains supersaturated.
Keep in mind, that this protein crystallization phase diagram is (just) a general framework. There are not that many phase diagrams of actual proteins available, actually. A nice reality check and a thorough introduction into the topic of protein crystallization phase diagrams can be found here:
Asherie, N. (2004)
Protein Crystallization and Phase Diagrams
Methods 34, 266-272