You have been working on your protein for some time, doing all kinds of kinetics characterization, looking at binding partners through pull downs or looking at its cellular localization. Basically, you’re doing cellular and molecular biology of your protein. Have you thought about trying to crystallize it? Aren’t you curious how it looks and how it’s structure affects its function? If you work on a protein which hasn’t yet been determined via crystallography/NMR, then read on!
What should I check before thinking toward protein crystallography?
Protein crystallography is one of those fields that luck plays ~50% of success and the rest is composed of good handling and brains. If you’re lucky (and experienced) you can get a determined structure two months after you obtain a positive sequence clone. No fiction, I have seen it happen.
Eager to read more? Here’s a list of the first things you should check before ordering crystallography screens from Hampton research:
- Is there any determined structure of your protein in the Protein Data Base (PDB)? Do a sequence search here.
- Is this protein predicted to be folded? Run a check at XtalPred meta server to get your bearings on many biochemical criteria which can affect the chances your protein can be crystallized. XtalPred also generates a list of homologues which their structure was determined.
- If you haven’t purified the protein yet, you should first do expression tests of the gene cloned into expression vectors (pET, pGEX, pMAL etc.). You should first assess which expression system is best suited for your protein. I recommend you visit the EMBL protein expression and purification facility website for a detail of the different steps for expression and purification. See additional posts about expression and purification posted by me.
- Do you have a pure protein sample? This is tricky, as people interpret “pure” as many shades of grey. Protein crystallographers regard a pure protein sample when visualizing the sample on SDS-PAGE post coomassie or silver stain which shows a single band of your protein (minimum of 40 microgram loaded on a single lane). Even if your samples contains a few faint bands it can be regarded as suitable for crystallography (see below):
- An additional and important consideration is that your protein should be homogenous. This is as important as purity level. A pure protein might have multiple oligomeric forms, which have a direct effect on its potential to form a crystal lattice. Thus, even if you get a really pure protein, you should perform a size exclusion chromatography or a Dynamic Light Scattering (DLS) experiment and evaluate the homogeneity level of your sample.
- How much resources have been invested in getting the protein sample to this purity level? This is a critical question because many times purification of a protein requires a special co-factor which can be expensive or it can be expressed at very low levels and it took much effort to get one microgram of pure sample. For clarification of the amount of purified protein which is required for crystallography, here is a quick estimate. An Initial screen of 96 conditions within one temperature setting and one protein concentration can require a minimum of 50 microlitters of five milligrams per ml of your protein (i.e. 250 microgram in total). And this is the flat minimum! For many biochemists this is the amount of protein they will consume in a whole PhD study. Furthermore, in many cases there is a batch-to-batch purification difference which is significant enough to lead to improve or worsen crystal growth. Thus it is recommended to purify as much protein as possible so it will last several rounds of screening and polishing of optimal crystal growth conditions.
- Do you have a crystallography lab/center which you can collaborate with or pay for service/devices? Mainstream crystallography requires a room or incubator which is temperature and humidity controlled and diffraction experiment requires an X-Ray generator (a minimum) or a synchrotron for obtaining improved diffraction data. Most research centers today have at least one crystallographer which have access to such facilities (and can also aid in your first experiments).
Do you qualify for all the above? If YES, then read my next post on how to prepare your protein for the challenge of protein crystallography!.