Protein Crystallisation Strategy

Now that your protein is stable and at an appropriate concentration, the rest of your challenge is focused on growing protein crystals which are good enough to allow x-ray diffraction studies. While temperature, drop size and plate architecture are limited by what is available in C3, the combination of chemicals to screen against is potentially infinite (read more about how we work with screens). We are asked “Which screen should I use?” more than any other question. The answer to this is dependent on how much protein you have, and how much you’re willing to use in the initial screening process.

What is a screen?

It refers to a kit of 96 unique chemical combinations. The chemical cocktails are often called ‘crystallants’ or ‘conditions’ or ‘wells’.

Where to Start?

Like all good strategies, ours is fluid. Our current first choice in screening has been distilled into the 1-Click Screening process, which takes the Shotgun screen at 8 and 20°C, then PACT and PS-Gradient screens at 20°C. If you can’t afford the 65-70 μL of protein required for this strategy (for the default drop size), then start with Shotgun on its own. Build on this with Shotgun using different protein concentrations and incubation temperatures, or try Shotgun and PACT. Use the sample calculator to figure out how many different experiments you can set up. If you’ve got ample protein and are happy to scry rather than screen, then try the C3 Set (768 unique conditions over 8 plates). This requires 130 μL of protein.

In summary, our suggestions are:

  1. 1-Click Screening (70 μL of sample)
  2. Shotgun at one temperature (25 μL of sample)
  3. Shotgun at two temperatures (40 μL of sample)
  4. Shotgun and PACT (40 μL of sample)
  5. The C3 Set (130 μL of sample)

The initial screening strategy should be a balance between the number of conditions, temperature and kinetics (drop ratio and size). Although it would be fantastic to scoop a diffraction quality crystal out of the initial screen, the real goal of initial screening is to find a starting point for optimisation (a hit). Remember that crystallisation screens are only one variable – temperature and drop ratio / drop size are others.

What is a hit?

A hit is a crystallisation condition (temperature, ratio) where there is some indication of crystallinity.

Next steps

If you’ve been really lucky, the initial screening will yield a clear hit result, something that looks obviously crystalline and fluoresces under UV light. If you’ve been really unlucky all the drops are clear (under concentrated) or all drops are precipitated (over concentrated), If not, then try to map out some commonalities in the behaviour of your protein in different chemicals.

  • Are precipitation events salt, pH, PEG dependent?
  • Which chemicals create light precipitate (maybe pursue) and which form heavy (avoid)?
  • What do the different PEG sizes and concentrations do to the protein?
  • Does it like / not like organics?
  • Is there some correlation between anion / cation classes and precipitation?
  • Which conditions are always clear (your protein is still in solution)?

In doing so you may be able to define some chemicals limits and choose a more specific screen (e.g. “low MW PEGS” or “sulfates”). If the initial trials yield no information, then it may be time to reassess the protein sample: look at alternative protein formulations, try a digest, throw in a known co-factor. If everything fails, then you may need to try altering the protein construct. If you have any clues as to which conditions to screen against – for example from the literature or previous experiments – use the C6 software to help find screens with the closest conditions. If you’ve got a hit and you’re sure they are protein crystals (or crystalline material), then there are four immediate paths available in C3 for optimisation.

NEXT: Optimisation