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 C3 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.

READ MORE: Screen Design in See3

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 a relatively cautious, but can be made less so by adding additional components. Booking a fine screening experiment is a three step process:

  1. You have to design the fine screen using See3. A logical name will be allocated to the design, e.g. GRID_<barcode>_<subwell>, you may rename this if you like.
  2. You must book time for that screen to be made using the TECAN resource on the booking site.
  3. The screen design only becomes available after we physically make it, so you could wait until this happens to book a nano-dispenser experiment, or select My Design Placeholder design to reserve the experiment slot, but remember to specify in the comments section of the booking wizard the name of your design.

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 additive screens, and the conditions are available at C6 (comparison of crystallisation conditions at C3). Booking a fine screen optimisation experiment is a three step process:

  1. Additive screens require a design to be made using See3. Click here for a summary of how to do this. When you save the design; you must leave the beginning of the design name as “Add_” otherwise it will not be recognised by the booking wizard.
  2. As there is no need to make a physical screen, the design should be immediately available for you to select in the batchload wizard when you make a booking.
  3. For each screen we require 10mL of base condition to be provided (clearly labelled). We can make it for you in C3, to do so you will need to book a 1-tube Tecan experiment

*Please note that it may be impossible to design an optimisation screen from an additive screen at C3. This is due to the fact that we may not have the chemical from the additive screen as a stock. Please email us if you have any queries regarding this.

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 which ever 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

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.

READ MORE: Matrix Seeding

Often crystals will grow with a morphology that makes them inappropriate to harvest, or they may be huge yet return low resolution structures. Waste not, want not: take the failed crystals and crush them up to form a seed stock and re-run the screen. Booking this experiment is a two step process:

  1. Make a seed stock with a solution of crystallant and crushed up crystals (instructions for making seed stock here).
  2. Use the booking wizard as normal, specifying in the comments section (on the wizard and on the web form) that you would like a seeding experiment, typically 50:40:10% protein:crystallant:seed-stock.

Try to make the seed-stock as close to the time of booking as possible, chances are the seed crystals are dissolving back to free protein in solution. It is good to use the dual subwells to include a control (non seeded) experiment in the same plate.

Drop modulation: changing the rates of vapour equilibration and component concentration within a crystallisation experiment.

If you’ve managed to grow many small and/or badly diffracting crystals, then altering the rate at which a crystallisation experiment comes to equilibrium and the final concentration of protein in a drop may increase the chance of growing a large and well-diffracting crystal. A simple optimisation that changes the kinetics would be identical plates at two temperatures, with drops of differing ratios. There are several methods that can easily modulate your experiment:

  • By changing the drop size you can vary the equilibration rate; big drops take longer to reach equilibrium.
  • Change the ratio of protein:crystallant will effect the concentration of protein and precipitant once equilibrium is reached. In addition drops with a high ratio of volatile components will reach equilibrium faster.
  • Changing the temperature with change the kinetics of crystal growth (colder = slower) and the vapour pressure of the experiment (keeps more stuff in the drop)
  • Setting up over an optimisation gradient (e.g. the Optimisation Gradient MkII Screen with NaCl, PEG 4000, CH3COONH4, (NH4)2SO4, CH3COONa) which will change the rate at which water is pulled out of the drop.

These variables can be all be changed within the booking-wizard. Total drop volumes should not exceed 1000nL, and we can safely incubate plates at 4C but these must be manually inspected. If you would like to work beyond these limits please discuss the possibilities with us.

Combination: combining two of the above techniques to optimise a hit.

To book a combined optimisation experiment, use the protocols for each step of the individual optimisations. If you want to try a more complex optimisation strategy, feel free to discuss it with one of the C3 scientists. We regularly test novel ideas for optimisation and are happy to develop new methodologies. It is not uncommon for crystallographers to complete the following combinations:

  • Additive with Seeding and/or Drop Modulation
  • Grid Screen with Seeding and/or Drop Modulation