In the previous post I have introduced non-crystallographers into evaluating whether their project is worthy of exploring in crystallography. Assuming you’re project is appropriate and you have the resources needed, read on for a primer on protein crystallography from protein to crystals.
Trial and error: Adapt your protein to crystallography world
You’re not the only one that needs to adapt to the protein crystallography world – your protein should too! You see, in protein crystallography the aim is to force billions of vibrating, moving and colliding protein molecules to pack into a crystal lattice in such a miraculous way that they will eventually form a solid creature, the crystal. For this miracle to happen the protein should be highly pure and contained within an environment which supports such delicate yet stable interactions. A 0.5 change in the pH value (corresponding to five fold change in proton equilibrium) is enough to inhibit any crystal formation. So, how do you know which buffer will be appropriate for crystal growth? Well, you don’t. This is the name of the game: trial and error. However, there are several starting points which you should aim for:
• Salt – for well behaving proteins (i.e. soluble at >5mg) you might want to limit the salt concentration in the buffer to 50-150mM (depending on the type of ion). Higher Salt concentrations can promote crystal formation in cases where it lead to dehydration of the protein which promotes protein-protein interactions. Of course, proteins should be kept at their solubility space and if higher salt concentration is needed so be it.
• Buffer & pH – Use of low concentration buffer above or below isoelectric point (pI)– since you can’t determine the exact pI of your protein, and since it can have a critical effect on the protein-protein interactions it is wise to enable your protein to move across the whole range of pH in the presence of the crystallization cocktail (see below). Yeah, it for sure will precipitate in a “bad” manner but it can also precipitate in a “good” manner (i.e. form a crystal). Thus, use as low buffer as possible such as 10-30mM.
• Additives – Remove any additives which are not required for the protein’s stability and solubility. This means, stabilizers, chelating agents, metal ions etc. as long as you KNOW these are a requirement.
• Protein concentration – Another tough question to answer. Since it is easier to dilute a protein solution rather than concentrate it I would recommend that upon completion of your purification scheme you should concentrate the protein solution to a concentration of 20mg/ml or higher, divide your protein solution into aliquots of 20-30 microliters, flash freeze it in liquid nitrogen and store it in -80 degrees Celsius. Why such small aliquots? That’s because once you’re thawed the protein solution you should not freeze it again (freeze-thaw cycles promote protein aggregation and denaturation).
Of course, all of the above are starting points; with time, you will familiarize with your protein in such a way that you will know which conditions are good for it and which are bad.
Next post I will discuss how to perform your first crystallization experiments!
Have any further questions? any further ideas?? Let me know!