Optimisation

When we optimise, we’re trying to change the conditions so that we increase the probability of growing crystals suitable for X-ray diffraction studies.

Take a look at what you’re optimising from (the hit condition) and think about how you can change this so that the experiment moves to the optimal location on a crystallisation phase diagram. Optimisation strategies are only limited by imagination, the availability of protein and suitable technology to set up the experiments. At BCC we offer a range of optimisation strategies; if you’re unsure of what to do and how to book it, or if you want your experiment to deviate from the standard protocols, please talk with us to help clarify information.

Fine Screening
Additive Screening
Seeding
Drop Modulation
Combination

Fine screening: step iteratively from a hit condition altering the ratio of chemical components.

There are two types of fine screen designs; Grid and Random. Grids are an evenly stepped series of conditions within user-defined limits, while Random picks points in –ve and +ve direction with random step sizes. Both methods are relatively cautious but can be made less so by adding additional components.

Additive screening: testing the effect of different chemicals on a hit condition.

For an additive screen, we take a ‘base condition’ – usually the best of a bad group of hits, and tweak it by adding a small amount of other chemicals. There are several available. There are a few different types of additive screens, and how they are set up depends on the cost of the additives, the behaviour of the additives and the robot capabilities. The most common is to mix 10% additive with the base condition and use this as crystallant in whichever ratio you decide, usually 50:50 protein:crystallant. If the additive is considered precious or difficult, we will only dispense it on the drop, with base condition being 100 per cent of the reservoir. The final drop will usually be 10:50:40% ratio additive:protein:base condition.

Additive mixed with reservoir and Additive mixed with Drop.
Reverse additive screen: The alternative additive method is to use a standard screen, say JCSG+, and a fixed additive. This method is useful if you possess an inhibitor or co-factor that is thought to positively interact with the protein, or find a hit chemical in one of the standard additive screens. Depending on the chemical nature of the additive, we make it by either mixing it with the reservoir or dispensing directly into the drop.

Seeding: increasing the probability of crystal growth by adding nucleation points.